Heterocyclic compounds and uses thereof

ABSTRACT

The present invention provides compounds, pharmaceutically acceptable compositions thereof, and methods of using the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional application of U.S. patentapplication Ser. No. 12/132,537, filed Jun. 3, 2008 and issued as U.S.Pat. No. 8,242,271 on Aug. 14, 2012, which claims priority to U.S.provisional patent application Ser 60/941,873, filed Jun. 4, 2007, andU.S. provisional patent application Ser. No. 60/972,048, filed Sep. 13,2007, the entirety of each of which is hereby incorporated herein byreference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compounds useful as inhibitors ofprotein kinases. The invention also provides pharmaceutically acceptablecompositions comprising compounds of the present invention and methodsof using said compositions in the treatment of various disorders.

BACKGROUND OF THE INVENTION

The search for new therapeutic agents has been greatly aided in recentyears by a better understanding of the structure of enzymes and otherbiomolecules associated with diseases. One important class of enzymesthat has been the subject of extensive study is protein kinases.

Protein kinases constitute a large family of structurally relatedenzymes that are responsible for the control of a variety of signaltransduction processes within the cell. Protein kinases are thought tohave evolved from a common ancestral gene due to the conservation oftheir structure and catalytic function. Almost all kinases contain asimilar 250-300 amino acid catalytic domain. The kinases may becategorized into families by the substrates they phosphorylate (e.g.,protein-tyrosine, protein-serine/threonine, lipids, etc.).

In general, protein kinases mediate intracellular signaling by effectinga phosphoryl transfer from a nucleoside triphosphate to a proteinacceptor that is involved in a signaling pathway. These phosphorylationevents act as molecular on/off switches that can modulate or regulatethe target protein biological function. These phosphorylation events areultimately triggered in response to a variety of extracellular and otherstimuli. Examples of such stimuli include environmental and chemicalstress signals (e.g., osmotic shock, heat shock, ultraviolet radiation,bacterial endotoxin, and H₂O₂), cytokines (e.g., interleukin-1 (IL-1)and tumor necrosis factor α (TNF-α)), and growth factors (e.g.,granulocyte macrophage-colony-stimulating factor (GM-CSF), andfibroblast growth factor (FGF)). An extracellular stimulus may affectone or more cellular responses related to cell growth, migration,differentiation, secretion of hormones, activation of transcriptionfactors, muscle contraction, glucose metabolism, control of proteinsynthesis, and regulation of the cell cycle.

Many diseases are associated with abnormal cellular responses triggeredby protein kinase-mediated events as described above. These diseasesinclude, but are not limited to, autoimmune diseases, inflammatorydiseases, bone diseases, metabolic diseases, neurological andneurodegenerative diseases, cancer, cardiovascular diseases, allergiesand asthma, Alzheimer's disease, and hormone-related diseases.Accordingly, there remains a need to find protein kinase inhibitorsuseful as therapeutic agents.

SUMMARY OF THE INVENTION

It has now been found that compounds of this invention, andpharmaceutically acceptable compositions thereof, are effective asinhibitors of one or more protein kinases. Such compounds have thegeneral formula I:

or a pharmaceutically acceptable salt thereof, wherein T, W, R^(a),R^(b), R^(c), R^(d), R¹, R², and R³ are as defined herein.

Compounds of the present invention, and pharmaceutically acceptablecompositions thereof, are useful for treating a variety of diseases,disorders or conditions, associated with abnormal cellular responsestriggered by protein kinase-mediated events. Such diseases, disorders,or conditions include those described herein.

Compounds provided by this invention are also useful for the study ofkinases in biological and pathological phenomena; the study ofintracellular signal transduction pathways mediated by such kinases; andthe comparative evaluation of new kinase inhibitors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the dose response inhibition of cell proliferation ofEOL-1 cells with reference compound and compound II-2.

FIG. 2 depicts the inhibition of PDGFR with reference compound andcompound II-2 in a “washout” experiment using EOL-1 cells.

FIG. 3 depicts the inhibition of c-KIT with compound III-14 in a“washout” experiment using GIST430 cells.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS 1. General Description ofCompounds of the Invention

In certain embodiments, the present invention provides a compound offormula I:

or a pharmaceutically acceptable salt thereof, wherein:T is —NHC(O)— or —C(O)NH—;W is CH or N;each of R^(a), R^(b), R^(c), and R^(d) is independently selected from R,OR, or halogen;each R is independently hydrogen, lower alkyl, or lower haloalkyl;R¹ is a warhead group;R² is selected from R, halogen, —N(R)C(O)OR, or 1-imidazoyl substitutedwith R, or:

-   -   R¹ and R² are taken together with their intervening atoms to        form a 5-7 membered saturated, partially unsaturated, or aryl        ring having 0-3 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, wherein said ring is substituted        with a warhead group and 0-3 groups independently selected from        oxo, halogen, CN, or C₁₋₆ aliphatic; and        R³ is selected from hydrogen, lower alkyl, or halogen.

In certain embodiments, the present invention provides a compound offormula II or III:

-   or a pharmaceutically acceptable salt thereof, wherein:-   each W is independently CH or N;-   each of R^(a), R^(b), R^(c), and R^(d) is independently selected    from R, OR, or halogen;-   each R is independently hydrogen, lower alkyl, or lower haloalkyl;-   each R¹ is independently a warhead group;-   each R² is independently selected from R, halogen, —N(R)C(O)OR, or    1-imidazoyl substituted with R, or:    -   R¹ and R² are taken together with their intervening atoms to        form a 5-7 membered saturated, partially unsaturated, or aryl        ring having 0-3 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, wherein said ring is substituted        with a warhead group and 0-3 groups independently selected from        oxo, halogen, CN, or C₁₋₆ aliphatic; and-   R³ is selected from hydrogen, lower alkyl, or halogen.

2. Compounds and Definitions

Compounds of this invention include those described generally above, andare further illustrated by the classes, subclasses, and speciesdisclosed herein. As used herein, the following definitions shall applyunless otherwise indicated. For purposes of this invention, the chemicalelements are identified in accordance with the Periodic Table of theElements, CAS version, Handbook of Chemistry and Physics, 75^(th) Ed.Additionally, general principles of organic chemistry are described in“Organic Chemistry”, Thomas Sorrell, University Science Books,Sausalito: 1999, and “March's Advanced Organic Chemistry”, 5^(th) Ed.,Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, theentire contents of which are hereby incorporated by reference.

As described herein, compounds of the invention may optionally besubstituted with one or more substituents, such as are illustratedgenerally above, or as exemplified by particular classes, subclasses,and species of the invention. It will be appreciated that the phrase“optionally substituted” is used interchangeably with the phrase“substituted or unsubstituted.” In general, the term “substituted”,whether preceded by the term “optionally” or not, refers to thereplacement of hydrogen radicals in a given structure with the radicalof a specified substituent. Unless otherwise indicated, an optionallysubstituted group may have a substituent at each substitutable positionof the group, and when more than one position in any given structure maybe substituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. Combinations of substituents envisioned by this invention arepreferably those that result in the formation of stable or chemicallyfeasible compounds. The term “stable”, as used herein, refers tocompounds that are not substantially altered when subjected toconditions to allow for their production, detection, and preferablytheir recovery, purification, and use for one or more of the purposesdisclosed herein. In some embodiments, a stable compound or chemicallyfeasible compound is one that is not substantially altered when kept ata temperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week.

The term “aliphatic” or “aliphatic group”, as used herein, means astraight-chain (i.e., unbranched) or branched, substituted orunsubstituted hydrocarbon chain that is completely saturated or thatcontains one or more units of unsaturation, or a monocyclic hydrocarbonor bicyclic hydrocarbon that is completely saturated or that containsone or more units of unsaturation, but which is not aromatic (alsoreferred to herein as “carbocycle” “cycloaliphatic” or “cycloalkyl”),that has a single point of attachment to the rest of the molecule.Unless otherwise specified, aliphatic groups contain 1-6 aliphaticcarbon atoms. In some embodiments, aliphatic groups contain 1-5aliphatic carbon atoms. In other embodiments, aliphatic groups contain1-4 aliphatic carbon atoms. In still other embodiments, aliphatic groupscontain 1-3 aliphatic carbon atoms, and in yet other embodimentsaliphatic groups contain 1-2 aliphatic carbon atoms. In someembodiments, “cycloaliphatic” (or “carbocycle” or “cycloalkyl”) refersto a monocyclic C₃-C₈ hydrocarbon or bicyclic C₈-C₁₀ hydrocarbon that iscompletely saturated or that contains one or more units of unsaturation,but which is not aromatic, that has a single point of attachment to therest of the molecule wherein any individual ring in said bicyclic ringsystem has 3-7 members. Suitable aliphatic groups include, but are notlimited to, linear or branched, substituted or unsubstituted alkyl,alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl,(cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The term “lower alkyl” refers to a C₁₋₄ straight or branched alkylgroup. Exemplary lower alkyl groups are methyl, ethyl, propyl,isopropyl, butyl, isobutyl, and tert-butyl.

The term “lower haloalkyl” refers to a C₁₋₄ straight or branched alkylgroup that is substituted with one or more halogen atoms.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen,phosphorus, or silicon (including, any oxidized form of nitrogen,sulfur, phosphorus, or silicon; the quaternized form of any basicnitrogen or; a substitutable nitrogen of a heterocyclic ring, forexample N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) orNR⁺ (as in N-substituted pyrrolidinyl)).

The term “unsaturated”, as used herein, means that a moiety has one ormore units of unsaturation.

As used herein, the term “bivalent C₁₋₈ [or C₁₋₆] saturated orunsaturated, straight or branched, hydrocarbon chain”, refers tobivalent alkylene, alkenylene, and alkynylene chains that are straightor branched as defined herein.

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH₂)_(n)—, wherein n is apositive integer, preferably from 1 to 6, from 1 to 4, from 1 to 3, from1 to 2, or from 2 to 3. A substituted alkylene chain is a polymethylenegroup in which one or more methylene hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

As used herein, the term “cyclopropylenyl” refers to a bivalentcyclopropyl group of the following structure:

The term “halogen” means F, Cl, Br, or I.

The term “aryl” used alone or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxyalkyl”, refers to monocyclic orbicyclic ring systems having a total of five to fourteen ring members,wherein at least one ring in the system is aromatic and wherein eachring in the system contains 3 to 7 ring members. The term “aryl” may beused interchangeably with the term “aryl ring”.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, Z and E double bond isomers,and Z and E conformational isomers. Therefore, single stereochemicalisomers as well as enantiomeric, diastereomeric, and geometric (orconformational) mixtures of the present compounds are within the scopeof the invention. Unless otherwise stated, all tautomeric forms of thecompounds of the invention are within the scope of the invention.Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, compounds having thepresent structures including the replacement of hydrogen by deuterium ortritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbonare within the scope of this invention. Such compounds are useful, forexample, as analytical tools, as probes in biological assays, or astherapeutic agents in accordance with the present invention. In someembodiments, the R¹ group of formula I comprises one or more deuteriumatoms.

As used herein, the term “irreversible” or “irreversible inhibitor”refers to an inhibitor (i.e. a compound) that is able to be covalentlybonded to a target protein kinase in a substantially non-reversiblemanner. That is, whereas a reversible inhibitor is able to bind to (butis generally unable to form a covalent bond) the target protein kinase,and therefore can become dissociated from the target protein kinase, anirreversible inhibitor will remain substantially bound to the targetprotein kinase once covalent bond formation has occurred. Irreversibleinhibitors usually display time dependency, whereby the degree ofinhibition increases with the time with which the inhibitor is incontact with the enzyme. Methods for identifying if a compound is actingas an irreversible inhibitor are known to one of ordinary skill in theart. Such methods include, but are not limited to, enzyme kineticanalysis of the inhibition profile of the compound with the proteinkinase target, the use of mass spectrometry of the protein drug targetmodified in the presence of the inhibitor compound, discontinuousexposure, also known as “washout,” experiments, and the use of labeling,such as radiolabelled inhibitor, to show covalent modification of theenzyme, as well as other methods known to one of skill in the art.

One of ordinary skill in the art will recognize that certain reactivefunctional groups can act as “warheads.” As used herein, the term“warhead” or “warhead group” refers to a functional group present on acompound of the present invention wherein that functional group iscapable of covalently binding to an amino acid residue (such ascysteine, lysine, histidine, or other residues capable of beingcovalently modified) present in the binding pocket of certain proteinkinases, thereby irreversibly inhibiting the protein kinase. Exemplaryreactive chemical functionalities useful as warheads include, but arenot limited to, acrylamides, α-,β-, and N-substituted acrylamides,vinylsulfonamides, vinylsulfones, epoxysulphones, allyl sulphones,epoxides, aza peptide epoxides, α,β-unsaturated carbonyl derivatives,α,β unsaturated alcohols, Michael acceptors with acyl lactam, acyloxazolidinone, and acyl urea functionalities, ester-derived Michaelacceptors with substituted alcohol groups, cis-α,β-unsaturated esters ortrans-α,β-unsaturated esters substituted at the α-position,amide-containing Michael acceptors, coumarins, cinnamates, andchalcones.

As used herein, the term “inhibitor” is defined as a compound that bindsto and/or inhibits the target protein kinase with measurable affinity.In certain embodiments, an inhibitor has an IC₅₀ and/or binding constantof less about 50 μM, less than about 1 μM, less than about 500 nM, lessthan about 100 nM, or less than about 10 nM.

The terms “measurable affinity” and “measurably inhibit,” as usedherein, means a measurable change in PDGFR (alpha or beta), cKit, KDR,or cFMS, activity between a sample comprising a compound of the presentinvention, or composition thereof, and a PDGFR (alpha or beta), cKit,KDR, or cFMS, and an equivalent sample comprising PDGFR (alpha or beta),cKit, KDR, or cFMS, in the absence of said compound, or compositionthereof.

3. Description of Exemplary Compounds

According to one aspect, the present invention provides a compound offormula II-a:

or a pharmaceutically acceptable salt thereof, wherein:W is CH or N;each of R^(a), R^(b), R^(c), and R^(d) is independently selected from R,OR, or halogen;each R is independently hydrogen, lower alkyl, or lower haloalkyl;R¹ is -L-Y, wherein:

-   -   L is a covalent bond or a bivalent C₁₋₈ saturated or        unsaturated, straight or branched, hydrocarbon chain, wherein        one, two, or three methylene units of L are optionally and        independently replaced by —NR—, —N(R)C(O)—, —C(O)N(R)—, —O—,        —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—,        or —C(═N₂)—;    -   Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo,        halogen, or CN, or a 3-10 membered monocyclic or bicyclic,        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, and wherein said ring is substituted with at 1-4 groups        independently selected from -Q-Z, oxo, halogen, CN, or C₁₋₆        aliphatic, wherein:        -   Q is a covalent bond or a bivalent C₁₋₆ saturated or            unsaturated, straight or branched, hydrocarbon chain,            wherein one or two methylene units of Q are optionally and            independently replaced by —NR—, —S—, —O—, —C(O)—, —SO—, or            —SO₂—; and        -   Z is hydrogen or C₁₋₆ aliphatic optionally substituted with            oxo, halogen, or CN;            R² is selected from R, halogen, —N(R)C(O)OR, or 1-imidazoyl            substituted with R, or:    -   R¹ and R² are taken together with their intervening atoms to        form a 5-7 membered saturated, partially unsaturated, or aryl        ring having 0-3 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, wherein said ring is substituted        with a warhead group, wherein the warhead group is -Q-Z, and        said ring is further substituted with 0-3 groups independently        selected from oxo, halogen, CN, or C₁₋₆ aliphatic; and        R³ is selected from hydrogen, lower alkyl, or halogen.

In certain embodiments, the W group is CH. In other embodiments, W is N.Thus, another embodiment of the present invention provides a compound ofeither of formulae II-b or II-c:

or a pharmaceutically acceptable salt thereof, wherein each of R^(a),R^(b), R^(c), R^(d), R¹, R², and R³ is as defined above and described inclasses and subclasses herein.

In certain embodiments, each of the R^(a), R^(b), R^(c), and R^(d)groups is hydrogen.

As defined generally above, R¹ is -L-Y, wherein L is a covalent bond ora bivalent C₁₋₈ saturated or unsaturated, straight or branched,hydrocarbon chain, wherein one, two, or three methylene units of L areoptionally and independently replaced by —NR—, —N(R)C(O)—, —C(O)N(R)—,—O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—,—N═N—, or —C(═N₂)—, and Y is hydrogen, C₁₋₆ aliphatic, or a 3-7 memberedsaturated, partially unsaturated, or aryl ring having 0-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, and whereinsaid ring is substituted with 1-4 groups independently selected fromoxo, halogen, CN, C₁₋₆ aliphatic, or -Q-Z.

In certain embodiments, L is a covalent bond.

In other embodiments, L is a bivalent C₁₋₈ saturated or unsaturated,straight or branched, hydrocarbon chain wherein at least one methyleneunit of L is replaced by —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, or—C(═N₂)—, and one or two additional methylene units of L are optionallyand independently replaced by —O—, —N(R)—, or —C(O)—. In someembodiments, L is a bivalent C₂₋₈ straight or branched, hydrocarbonchain wherein L has at least one double bond and at least one methyleneunit of L is replaced by —NRC(O)—, —C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—,—S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, or —C(═N₂)—, and one or two additionalmethylene units of L are optionally and independently replaced bycyclopropylene, —O—, —N(R)—, or —C(O)—. In still other embodiments, L isa bivalent C₁₋₈, straight or branched, alkylene chain, wherein at leastone methylene unit of L is replaced by —S—, —S(O)—, —SO₂—, —C(═S)—,—C(═NR)—, or —C(═N₂)—, and one or two additional methylene units of Lare optionally and independently replaced by —O—, —N(R)—, or —C(O)—. Insome embodiments, L is a bivalent C₁₋₈, straight or branched, alkylenechain, wherein at least one methylene unit of L is replaced by —C(═N₂)—,and one or two additional methylene units of L are optionally andindependently replaced by —NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—,—C(═S)—, —C(═NR)—, —O—, —N(R)—, or —C(O)—.

In certain embodiments, L is a bivalent C₁₋₈ saturated or unsaturated,straight or branched, hydrocarbon chain. In certain embodiments, L is—CH₂—.

In some embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one triple bond. In certainembodiments, L is a bivalent C₂₋₈ straight or branched, hydrocarbonchain wherein L has at least one triple bond and one or two additionalmethylene units of L are optionally and independently replaced by—NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, —O—, —N(R)—,or —C(O)—. In some embodiments, L has at least one triple bond and atleast one methylene unit of L is replaced by —N(R)—, —N(R)C(O)—, or —O—.

Exemplary L groups include —C≡C—, —C≡CCH₂N(isopropyl)-,—NHC(O)C≡CCH₂CH₂—, —CH₂—C≡C—CH₂—, and —C≡CCH₂O—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein L has at least one double bond and at leastone methylene unit of L is replaced by cyclopropylene, —NRC(O)—,—C(O)NR—, —N(R)SO₂—, —SO₂N(R)—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—,or —C(═N₂)—, and one or two additional methylene units of L areoptionally and independently replaced by —O—, —N(R)—, or —C(O)—.

Exemplary L groups include —CH₂—, —NH—, —CH₂NH—, —NHCH₂—, —NHC(O)—,—NHC(O)CH₂OC(O)—, —NHC(O)CH═CH—, —CH₂NHC(O)—, —NHC(O)CH═CHCH₂N(CH₃)—,—NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂—, —NHSO₂CH₂—, —NHSO₂CH═CH—,—NHSO₂CH═CHCH₂—, —NHC(O)(C═N₂)—, —NHC(O)(C═N₂)C(O)—, —NHC(O)CH₂OC(O)—,—SO₂NH—, —NHC(O)CH═CHCH₂N(CH₃)—, and —NHC(O)CH═CHCH₂O—.

As described above, in certain embodiments, L is a bivalent C₂₋₈straight or branched, hydrocarbon chain wherein L has at least onedouble bond. One of ordinary skill in the art will recognize that such adouble bond may exist within the hydrocarbon chain backbone or may be“exo” to the backbone chain and thus forming an alkylidene group. By wayof example, such an L group having an alkylidene branched chain includes—CH₂C(═CH₂)CH₂—. Thus, in some embodiments, L is a bivalent C₂₋₈straight or branched, hydrocarbon chain wherein L has at least onealkylidenyl double bond. Exemplary L groups include —NHC(O)C(═CH₂)CH₂—.

In certain embodiments, L is a bivalent C₂₋₈ straight or branched,hydrocarbon chain wherein one methylene unit of L is replaced bycyclopropylene and one or two additional methylene units of L areindependently replaced by —C(O)—, —NRC(O)—, —C(O)NR—, —N(R)SO₂—, or—SO₂N(R)—. Exemplary L groups include —NHC(O)-cyclopropylene-SO₂— and—NHC(O)-cyclopropylene-.

In certain embodiments, Y is hydrogen.

In certain embodiments, Y is C₁₋₆ aliphatic optionally substituted withoxo, halogen, or CN. In other embodiments, Y is C₁₋₆ alkyl. In someembodiments, Y is C₂₋₆ alkenyl. In other embodiments, Y is C₂₋₄ alkynyl.In certain embodiments, Y is cyclopropyl optionally substituted with —CNor —NO₂. In some embodiments, Y is cyclopropenyl or cyclobutenyl.

In certain embodiments, Y is a 3-10 membered monocyclic or bicyclic,saturated, partially unsaturated, or aryl ring having 0-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur, and whereinsaid ring is substituted with 1-4 groups independently selected from-Q-Z, oxo, halogen, CN, or C₁₋₆ aliphatic.

In some embodiments, Y is phenyl or pyridyl.

In certain embodiments, Y is a saturated 3-6 membered monocyclic ringhaving 1-3 heteroatoms independently selected from nitrogen, oxygen, orsulfur, wherein Y is substituted as defined above. In some embodiments,Y is a saturated 3-4 membered monocyclic ring having 1 heteroatomselected from oxygen or nitrogen. Exemplary rings are epoxide andoxetane rings. In other embodiments, Y is a saturated 5-6 memberedmonocyclic ring having 1-2 heteroatom selected from oxygen or nitrogen.Such rings include piperidine and pyrrolidine.

In other embodiments, Y is a 5 membered partially unsaturated or arylring having 1-3 heteroatoms independently selected from nitrogen,oxygen, or sulfur, wherein Y is substituted as defined above. In someembodiments, Y is a 5 membered partially unsaturated or aryl ring having1-3 heteroatoms independently selected from nitrogen, oxygen, andsulfur. Exemplary rings are thiadiazole, oxazole, oxadiazole, and2,5-dihydro-1H-pyrrole.

In certain embodiments, Y is an 8-10 membered bicyclic, saturated,partially unsaturated, or aryl ring having 0-3 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, wherein Y is substituted asdefined above. According to another aspect, Y is a 9-10 memberedbicyclic, partially unsaturated, or aryl ring having 1-3 heteroatomsindependently selected from nitrogen, oxygen, or sulfur. Exemplarybicyclic rings include 2,3-dihydrobenzo[d]isothiazole.

As defined generally above, when Y is a ring, said ring is substitutedwith 1-4 groups independently selected from -Q-Z, oxo, NO₂, halogen, CN,or C₁₋₆ aliphatic. In certain embodiments, Q is a bivalent C₁₋₆saturated or unsaturated, straight or branched, hydrocarbon chain,wherein one or two methylene units of Q are optionally and independentlyreplaced by —NR—, —NRC(O)—, —C(O)NR—, —S—, —O—, —C(O)—, —SO—, or —SO₂—.In other embodiments, Q is a bivalent C₂₋₆ straight or branched,hydrocarbon chain having at least one double bond, wherein one or twomethylene units of Q are optionally and independently replaced by —NR—,—NRC(O)—, —C(O)NR—, —S—, —O—, —C(O)—, —SO—, or —SO₂—. In someembodiments, -Q-Z is —NHC(O)CH═CH₂ or —C(O)CH═CH₂. In certainembodiments, the Y ring is substituted with at least one group selectedfrom oxo, fluoro, chloro, —NHC(O)CH═CH₂, —C(O)CH═CH₂, NO₂, —C(O)OEt, orCN.

In certain embodiments, Y is selected from those set forth in Table 1,below, wherein each wavy line indicates the point of attachment to therest of the molecule.

TABLE 1 Exemplary Y groups:

i

ii

iii

iv

v

vi

vii

viii

ix

x

xi

xii

xiii

xiv

xv

xvi

xvii

xviii

xix

xx

xxi

xxii

xxiii

xxiv

xxv

xxvi

xxvii

xxviii

xxix

xxx

xxxi

xxxii

xxxiii

xxxiv

xxxv

xxxvi

xxxvii

xxxviii

xxxix

xl

xli

xlii

xliii

xliv

xlv

xlvi

xlvii

xlviii

xlix

l

li

lii

liii

liv

lv

lvi

lvii

lviii

lix

lx

lxi

lxii

lxiii

lxiv

lxv

lxvi

lxvii

lxviii

lxix

lxx

lxxi

lxxii

lxxiii

lxxiv

lxxv

lxxvi

lxxvii

lxxviii

lxxix

lxxx

lxxxi

lxxxii

lxxxiii

lxxxiv

lxxxv

lxxxvi

lxxxvii

lxxxviii

lxxxix

xc

xci

xcii

xciii

xciv

xcv

xcvi

xcvii

xcviii

xcix

c

ci

cii

ciii

civ

cv

cvi

cvii

cviii

cix

In certain embodiments, Y is other than maleimide when L is —CH₂—. Inother embodiments, Y is other then epoxide when L is —CH₂—.

In certain embodiments, R¹ is selected from those set forth in Table 2,below, wherein each wavy line indicates the point of attachment to therest of the molecule.

TABLE 2 Exemplary R¹ Groups

i

ii

iii

iv

v

vi

vii

viii

ix

x

xi

xii

xiii

xiv

xv

xvi

xvii

xviii

xix

xx

xxi

xxii

xxiii

xiv

xv

xxvi

xxvii

xxviii

xxix

xxx

xxxi

xxxii

xxxiii

xxxiv

xxxv

xxxvi

xxxvii

xxxviii

xxxix

xl

xli

xlii

xliii

xliv

xlv

xlvi

xlvii

xlviii

xlix

l

li

lii

liii

liv

lv

lvi

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lix

lx

lxi

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lxix

lxx

lxxi

lxxii

lxxiii

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lxxviii

lxxix

lxxx

lxxxi

lxxxii

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lxxxvi

lxxxvii

lxxxviii

lxxxix

xc

xci

xcii

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xcix

c

ci

cii

ciii

civ

cv

cvi

cvii

cviii

cix

cx

cxi

cxii

cxiii

cxiv

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cxviii

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cxx

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cxxii

cxxiii

cxxiv

cxxv

cxxvi

cxxvii

cxxviii

In certain embodiments, R¹ is selected from —NHC(O)CH═CH₂ or—CH₂NHC(O)CH═CH₂.

As defined generally above, R¹ is a warhead group, or, when R¹ and R²form a ring, then -Q-Z is a warhead group. Without wishing to be boundby any particular theory, it is believed that such R¹ groups, i.e.warhead groups, are particularly suitable for covalently binding to akey cysteine residue in the binding domain of certain protein kinases.Protein kinases having a cysteine residue in the binding domain areknown to one of ordinary skill in the art and include PDGFR (alpha andbeta), cKit, KDR, and cFMS. In certain embodiments, compounds of thepresent invention have a warhead group characterized in that inventivecompounds target one or more of the following cysteine residues:

KDR SRK C IHRDLA (C1024; http://www.ebi.uniprot.org/entry/P35968) cKITAFLASKN C IH (C788; http://www.ebi.uniprot.org/entry/P10721) PDGFR-α SKNC VHRDLA (C814; http://www.ebi.uniprot.org/entry/P16234) PDGFR-β N CVHRDLAAR (C822; http://www.ebi.uniprot.org/entry/P09619) cFMS SKN CIHRDVA (C774; http://www.ebi.uniprot.org/entry/P07333)

Thus, in some embodiments, R¹ is characterized in that the -L-Y moietyis capable of covalently binding to a cysteine residue therebyirreversibly inhibiting the enzyme. One of ordinary skill in the artwill recognize that a variety of warhead groups, as defined herein, aresuitable for such covalent bonding. Such R¹ groups include, but are notlimited to, those described herein and depicted in Table 2, infra.

According to another aspect, the present invention provides a conjugatecomprising any of PDGFR-α, PDGFR-β, c-KIT, cFMS, or KDR, or a mutantthereof, covalently bonded to an inhibitor at Cys814, Cys822, Cys788,Cys774, or Cys 1024, respectively. In some embodiments, the inhibitor iscovalently bonded via a linker moiety.

In certain embodiments, the present invention provides a conjugate ofthe formula Cys814-linker-inhibitor moiety, Cys822-linker-inhibitormoiety, Cys788-linker-inhibitor moiety, Cys774-linker-inhibitor moiety,or Cys1024-linker-inhibitor moiety, wherein the Cys814 is of PDGFR-α;the Cys822 is of PDGFR-β; the Cys788 is of c-KIT; the Cys774 is of cFMSand the Cys1024 is of KDR. One of ordinary skill in the art willrecognize that the “linker” group corresponds to an -L-Y warhead groupas described herein. Accordingly, in certain embodiments, the linkergroup is as defined for -L-Y was defined above and described in classesand subclasses herein. It will be appreciated, however, that the linkergroup is bivalent and, therefore, the corresponding -L-Y group is alsointended to be bivalent resulting from the reaction of the warhead withthe targeted cysteine.

In certain embodiments, the inhibitor moiety is a compound of formula A:

wherein each of the R², R³, R^(a), R^(b), R^(c), R^(d), and W groups offormula A is as defined for formula I above and described in classes andsubclasses herein. Thus, in certain embodiments, the present inventionprovides a conjugate of any of formulae:

wherein each of the R², R³, R^(a), R^(b), R^(c), R^(d), and W groups offormula A is as defined for formula I above and described in classes andsubclasses herein and wherein the Cys814 is of PDGFR-α; the Cys822 is ofPDGFR-β; the Cys788 is of c-KIT; the Cys774 is of cFMS1 and the Cys1024is of KDR.

As defined generally above, R² is selected from R, halogen, —N(R)C(O)OR,or 1-imidazoyl substituted with R, or R¹ and R² are taken together withtheir intervening atoms to form a 5-7 membered saturated, partiallyunsaturated, or aryl ring having 0-3 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, wherein said ring is substituted with1-3 groups independently selected from oxo, halogen, CN, C₁₋₆ aliphatic,or -Q-Z.

In certain embodiments, R² is selected from R, halogen, —N(R)C(O)OR, or1-imidazoyl substituted with R. In some embodiments, R² is halogen. Inother embodiments, R² is R, wherein R is hydrogen. In still otherembodiments, R² is R, wherein R is lower alkyl or lower haloalkyl.Exemplary R² groups include methyl, ethyl, trifluoromethyl, chloro,bromo, fluoro, and iodo. In certain embodiments, R² is trifluoromethyl.

According to some aspects, R¹ and R² are taken together with theirintervening atoms to form a 5-7 membered saturated, partiallyunsaturated, or aryl ring having 0-3 heteroatoms independently selectedfrom nitrogen, oxygen, or sulfur, wherein said ring is substituted with1-3 groups independently selected from oxo, halogen, CN, C₁₋₆ aliphatic,or -Q-Z. In certain embodiments, R¹ and R² are taken together with theirintervening atoms to form, together with the phenyl ring fused thereto,a naphthyl, tetrahydroquinoline, indoline, isoindoline, 1H-indole, ortetrahydroisoquinoline ring. In certain embodiments, R¹ and R² are takentogether with their intervening atoms to form a naphthyl ringsubstituted with -Q-Z.

In certain embodiments, the ring formed by R¹ and R² is substituted with-Q-Z. In certain embodiments, the ring formed by R¹ and R² issubstituted with —NHC(O)CH═CH₂, —C(O)CH═CH₂, or —CH₂N(CH₃)₂. In someembodiments, R¹ and R² are taken together with their intervening atomsto form a naphthyl ring substituted with —NHC(O)CH═CH₂.

As defined generally above, R³ is selected from hydrogen, lower alkyl,or halogen. In certain embodiments, R³ is lower alkyl. In someembodiments, R³ is methyl.

According to another aspect, the present invention provides a compoundof formula III-a:

or a pharmaceutically acceptable salt thereof, wherein:W is CH or N;each of R^(a), R^(b), R^(c), and R^(d) is independently selected from R,OR, or halogen;each R is independently hydrogen, lower alkyl, or lower haloalkyl;R¹ is -L-Y, wherein:

-   -   L is a covalent bond or a bivalent C₁₋₈ saturated or        unsaturated, straight or branched, hydrocarbon chain, wherein        one, two, or three methylene units of L are optionally and        independently replaced by —NR—, —N(R)C(O)—, —C(O)N(R)—, —O—,        —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO—, —SO₂—, —C(═S)—, —C(═NR)—,        or —C(═N₂)—;    -   Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo,        halogen, or CN, or a 3-10 membered monocyclic or bicyclic,        saturated, partially unsaturated, or aryl ring having 0-3        heteroatoms independently selected from nitrogen, oxygen, or        sulfur, and wherein said ring is substituted with at 1-4 groups        independently selected from -Q-Z, oxo, halogen, CN, or C₁₋₆        aliphatic, wherein:        -   Q is a covalent bond or a bivalent C₁₋₆ saturated or            unsaturated, straight or branched, hydrocarbon chain,            wherein one or two methylene units of Q are optionally and            independently replaced by —NR—, —S—, —O—, —C(O)—, —SO—, or            —SO₂—; and        -   Z is hydrogen or C₁₋₆ aliphatic optionally substituted with            oxo, halogen, or CN;            R² is selected from R, halogen, —N(R)C(O)OR, or 1-imidazoyl            substituted with R, or:    -   R¹ and R² are taken together with their intervening atoms to        form a 5-7 membered saturated, partially unsaturated, or aryl        ring having 0-3 heteroatoms independently selected from        nitrogen, oxygen, or sulfur, wherein said ring is substituted        with a warhead group, wherein the warhead group is -Q-Z, and        said ring is further substituted with 0-3 groups independently        selected from oxo, halogen, CN, or C₁₋₆ aliphatic; and        R³ is selected from hydrogen, lower alkyl, or halogen.

In certain embodiments, each of the W, R^(a), R^(b), R^(c), R^(d), R¹,R², and R³ groups of formula III-a is as defined above and described inclasses and subclasses herein.

In certain embodiments, the R³ group of formula III-a is lower alkyl.

As described herein, in certain embodiments, the W group of formulaIII-a is CH and in other embodiments, W is N. Thus, another embodimentof the present invention provides a compound of either of formulae III-bor III-c:

or a pharmaceutically acceptable salt thereof, wherein each of R^(a),R^(b), R^(c), R^(d), R¹, R², and R³ is as defined above and described inclasses and subclasses herein.

Exemplary compounds of formula II are set forth in Table 3 below.

TABLE 3 Exemplary Compounds of Formula II

II-1

II-2

II-3

II-4

II-5

II-6

II-7

II-8

II-9

II-10

II-11

II-12

II-13

II-14

II-15

II-16

II-17

II-18

II-19

II-20

II-21

II-22

II-23

II-24

II-25

II-26

II-27

II-28

II-29

II-30

II-31

II-32

II-33

II-34

II-35

II-36

II-37

II-38

II-39

II-40

II-41

II-42

II-43

II-44

II-45

II-46

II-47

II-48

II-49

II-50

II-51

II-52

II-53

II-54

II-55

In certain embodiments, the present invention provides any compounddepicted in Table 3, above, or a pharmaceutically acceptable saltthereof.

Exemplary compounds of formula III are set forth in Table 4, below.

TABLE 4 Exemplary Compounds of Formula III

III-1

III-2

III-3

III-4

III-5

III-6

III-7

III-8

III-9

III-10

III-11

III-12

III-13

III-14

III-15

III-16

III-17

III-18

III-19

In certain embodiments, the present invention provides any compounddepicted in Table 4, above, or a pharmaceutically acceptable saltthereof.

4. Uses, Formulation and Administration

Pharmaceutically Acceptable Compositions

According to another embodiment, the invention provides a compositioncomprising a compound of this invention or a pharmaceutically acceptablederivative thereof and a pharmaceutically acceptable carrier, adjuvant,or vehicle. The amount of compound in compositions of this invention issuch that is effective to measurably inhibit a protein kinase,particularly PDGFR (alpha and beta), cKit, KDR, or cFMS, in a biologicalsample or in a patient. In certain embodiments, the amount of compoundin compositions of this invention is such that is effective tomeasurably inhibit a protein kinase, particularly PDGFR (alpha and beta)and/or cKit, in a biological sample or in a patient. Preferably acomposition of this invention is formulated for administration to apatient in need of such composition. Most preferably, a composition ofthis invention is formulated for oral administration to a patient.

The term “patient”, as used herein, means an animal, preferably amammal, and most preferably a human.

The term “pharmaceutically acceptable carrier, adjuvant, or vehicle”refers to a non-toxic carrier, adjuvant, or vehicle that does notdestroy the pharmacological activity of the compound with which it isformulated. Pharmaceutically acceptable carriers, adjuvants or vehiclesthat may be used in the compositions of this invention include, but arenot limited to, ion exchangers, alumina, aluminum stearate, lecithin,serum proteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, sodium carboxymethylcellulose,polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,polyethylene glycol and wool fat.

A “pharmaceutically acceptable derivative” means any non-toxic salt,ester, salt of an ester or other derivative of a compound of thisinvention that, upon administration to a recipient, is capable ofproviding, either directly or indirectly, a compound of this inventionor an inhibitorily active metabolite or residue thereof.

As used herein, the term “inhibitorily active metabolite or residuethereof” means that a metabolite or residue thereof is also an inhibitorof PDGFR (alpha and beta), cKit, KDR, or cFMS.

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeacetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN+(C₁₋₄ alkyl)₄ salts. This invention also envisions the quaternizationof any basic nitrogen-containing groups of the compounds disclosedherein. Water or oil-soluble or dispersible products may be obtained bysuch quaternization.

Compositions of the present invention may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term “parenteral”as used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional and intracranial injection or infusiontechniques. Preferably, the compositions are administered orally,intraperitoneally or intravenously. Sterile injectable forms of thecompositions of this invention may be aqueous or oleaginous suspension.These suspensions may be formulated according to techniques known in theart using suitable dispersing or wetting agents and suspending agents.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium.

For this purpose, any bland fixed oil may be employed includingsynthetic mono- or di-glycerides. Fatty acids, such as oleic acid andits glyceride derivatives are useful in the preparation of injectables,as are natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, such as carboxymethyl cellulose or similar dispersingagents that are commonly used in the formulation of pharmaceuticallyacceptable dosage forms including emulsions and suspensions. Othercommonly used surfactants, such as Tweens, Spans and other emulsifyingagents or bioavailability enhancers which are commonly used in themanufacture of pharmaceutically acceptable solid, liquid, or otherdosage forms may also be used for the purposes of formulation.

Pharmaceutically acceptable compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

Alternatively, pharmaceutically acceptable compositions of thisinvention may be administered in the form of suppositories for rectaladministration. These can be prepared by mixing the agent with asuitable non-irritating excipient that is solid at room temperature butliquid at rectal temperature and therefore will melt in the rectum torelease the drug. Such materials include cocoa butter, beeswax andpolyethylene glycols.

Pharmaceutically acceptable compositions of this invention may also beadministered topically, especially when the target of treatment includesareas or organs readily accessible by topical application, includingdiseases of the eye, the skin, or the lower intestinal tract. Suitabletopical formulations are readily prepared for each of these areas ororgans.

Topical application for the lower intestinal tract can be effected in arectal suppository formulation (see above) or in a suitable enemaformulation. Topically-transdermal patches may also be used.

For topical applications, provided pharmaceutically acceptablecompositions may be formulated in a suitable ointment containing theactive component suspended or dissolved in one or more carriers.Carriers for topical administration of compounds of this inventioninclude, but are not limited to, mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, providedpharmaceutically acceptable compositions can be formulated in a suitablelotion or cream containing the active components suspended or dissolvedin one or more pharmaceutically acceptable carriers. Suitable carriersinclude, but are not limited to, mineral oil, sorbitan monostearate,polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol and water.

For ophthalmic use, provided pharmaceutically acceptable compositionsmay be formulated as micronized suspensions in isotonic, pH adjustedsterile saline, or, preferably, as solutions in isotonic, pH adjustedsterile saline, either with or without a preservative such asbenzylalkonium chloride. Alternatively, for ophthalmic uses, thepharmaceutically acceptable compositions may be formulated in anointment such as petrolatum.

Pharmaceutically acceptable compositions of this invention may also beadministered by nasal aerosol or inhalation. Such compositions areprepared according to techniques well-known in the art of pharmaceuticalformulation and may be prepared as solutions in saline, employing benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, fluorocarbons, and/or other conventional solubilizingor dispersing agents.

Most preferably, pharmaceutically acceptable compositions of thisinvention are formulated for oral administration.

The amount of compounds of the present invention that may be combinedwith the carrier materials to produce a composition in a single dosageform will vary depending upon the host treated, the particular mode ofadministration. Preferably, provided compositions should be formulatedso that a dosage of between 0.01-100 mg/kg body weight/day of theinhibitor can be administered to a patient receiving these compositions.

It should also be understood that a specific dosage and treatmentregimen for any particular patient will depend upon a variety offactors, including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,rate of excretion, drug combination, and the judgment of the treatingphysician and the severity of the particular disease being treated. Theamount of a compound of the present invention in the composition willalso depend upon the particular compound in the composition.

Uses of Compounds and Pharmaceutically Acceptable Compositions

Compounds and compositions described herein are generally useful for theinhibition of protein kinase activity of one or more enzymes. Furtherinformation relating to kinase structure, function and their role indisease or disease symptoms is available at the Protein Kinase Resourcewebsite (http://kinases.sdsc.edu/html/index.shtml).

Drug resistance is emerging as a significant challenge for targetedtherapies. For example, drug resistance has been reported for Gleevec®and Iressa®, as well as several other kinase inhibitors in development.In addition, drug resistance has been reported for the cKit and PDGFRreceptors. It has been reported that irreversible inhibitors may beeffective against drug resistant forms of protein kinases (Kwak, E. L.,R. Sordella, et al. (2005). “Irreversible inhibitors of the EGF receptormay circumvent acquired resistance to gefitinib.” PNAS 102(21):7665-7670.) Without wishing to be bound by any particular theory, it isbelieved that compounds of the present invention may be effectiveinhibitors of drug resistant forms of protein kinases.

As used herein, the term “clinical drug resistance” refers to the lossof susceptibility of a drug target to drug treatment as a consequence ofmutations in the drug target

As used herein, the term “resistance” refers to changes in the wild-typenucleic acid sequence coding a target protein, and/or the proteinsequence of the target, which changes decrease or abolish the inhibitoryeffect of the inhibitor on the target protein.

Examples of kinases that are inhibited by the compounds and compositionsdescribed herein and against which the methods described herein areuseful include PDGFR (alpha and beta), cKit, KDR, and cFMS.

The activity of a compound utilized in this invention as an inhibitor ofPDGFR (alpha and beta), cKit, KDR, or cFMS, may be assayed in vitro, invivo or in a cell line. In vitro assays include assays that determineinhibition of either the phosphorylation activity and/or the subsequentfunctional consequences, or ATPase activity of activated PDGFR (alphaand beta), cKit, KDR, or cFMS. Alternate in vitro assays quantitate theability of the inhibitor to bind to PDGFR (alpha and beta), cKit, KDR,or cFMS. Inhibitor binding may be measured by radiolabelling theinhibitor prior to binding, isolating the inhibitor/PDGFR,inhibitor/cKit, inhibitor/KDR, or inhibitor/cFMS complex and determiningthe amount of radiolabel bound. Alternatively, inhibitor binding may bedetermined by running a competition experiment where new inhibitors areincubated with PDGFR (alpha and beta), cKit, KDR, or cFMS bound to knownradioligands. Detailed conditions for assaying a compound utilized inthis invention as an inhibitor of PDGFR (alpha and beta), cKit, KDR, orcFMS are set forth in the Examples below.

Tyrosine kinases are a class of enzymes that mediate intracellularsignal transduction pathways. Abnormal activity of these kinases hasbeen shown to contribute to cell proliferation, carcinogenesis and celldifferentiation. Thus, agents that modulate the activity of tyrosinekinases are useful for preventing and treating proliferative diseasesassociated with these enzymes.

Platelet-derived growth factor receptor (PDGFR) is an important targetin tumor cell proliferation, cell migration, and angiogenesis, and maymediate the high interstitial fluid pressure (IFP) of tumors. PDGFRsignaling pathways have been implicated in the development and growth ofsolid tumors. Blockade of PDGFR receptors has been shown to inhibitangiogenesis, tumor vascular maturation and maintenance, and tumor cellproliferation leading to tumor regression.

Additionally, PDGFR signals induce expression of proangiogenic signals(including VEGF) in endothelial cells, further stimulating tumorangiogenesis. PDGFR is essential for regulating the proliferation andmigration of pericytes to the tumor vessel in angiogenesis. Pericytesare smooth vascular muscle cells that, with the help of endothelialcells, provide a structure around which new blood vessels may be formed.The expression of PDGFR on pericytes is a key event involved in thematuration and survival of tumor vasculature. Additionally, emergingdata suggest that PDGF receptors mediate the high IFP of tumors, whichhas been shown to be a barrier to the efficient uptake ofchemotherapeutic drugs.

PDGF-receptor (PDGFR) has two subunits—PDGFR-α and PDGRR-β, which canform homo or heterodimers upon ligand binding. There are several PDGFligands: AB, BB, CC and DD. PDGFR is expressed on early stem cells, mastcells, myeloid cells, mesenchymal cells and smooth muscle cells. OnlyPDGFR-β has been implicated in myeloid leukemias, usually as atranslocation partner with Tel, Huntingtin interacting protein (HIP1) orRabaptin. Recently it was shown that activation mutations in PDGFR-αkinase domain play an important role in gastrointestinal stromal tumors(GIST).

According to another embodiment, the invention provides a method fortreating or lessening the severity of a PDGFR-mediated disease orcondition in a patient comprising the step of administering to saidpatient a composition according to the present invention.

As used herein, the terms “treatment,” “treat,” and “treating” refer toreversing, alleviating, delaying the onset of, or inhibiting theprogress of a disease or disorder, or one or more symptoms thereof, asdescribed herein. In some embodiments, treatment may be administeredafter one or more symptoms have developed. In other embodiments,treatment may be administered in the absence of symptoms. For example,treatment may be administered to a susceptible individual prior to theonset of symptoms (e.g., in light of a history of symptoms and/or inlight of genetic or other susceptibility factors). Treatment may also becontinued after symptoms have resolved, for example to prevent or delaytheir recurrence.

The term “PDGFR-mediated disease” or “condition”, as used herein meansany disease or other deleterious condition in which PDGFR is known toplay a role. Accordingly, another embodiment of the present inventionrelates to treating or lessening the severity of one or more diseases inwhich PDGFR is known to play a role. Specifically, the present inventionrelates to a method of treating or lessening the severity of a diseaseor condition selected from a proliferative disorder, wherein said methodcomprises administering to a patient in need thereof a compositionaccording to the present invention.

In certain embodiments, the present invention provides a method forinhibiting the growth of blood vessels. Such a method is useful fortreating disorders associated with deregulated angiogenesis, such asdiseases caused by ocular neovascularisation, including retinopathies,such as diabetic retinopathy or age-related macula degeneration,psoriasis, haemangioblastoma, such as haemangioma, mesangial cellproliferative disorders, such as chronic or acute renal diseases, e.g.diabetic nephropathy, malignant nephrosclerosis, thromboticmicroangiopathy syndromes or transplant rejection. Such disorders alsoinclude inflammatory renal disease, such as glomerulonephritis,mesangioproliferative glomerulonephritis, haemolytic-uraemic syndrome,diabetic nephropathy, hypertensive nephrosclerosis, atheroma, arterialrestenosis, autoimmune diseases, diabetes, endometriosis, chronicasthma, and neoplastic diseases (solid tumors, but also leukemias andother “liquid tumors”), such as breast cancer, cancer of the colon, lungcancer (e.g., small-cell lung cancer), cancer of the prostate orKaposi's sarcoma. In some embodiments, the present invention provides amethod of inhibiting the growth of tumours, including preventing themetastatic spread of tumors and the growth of micrometastases. Incertain embodiments, the present invention provides a method fortreating a leukemia.

According to another embodiment, the present invention relates to amethod of treating the growth or metastasis of tumor/cancer cells; andcarcinomas (e.g, squamous cell carcinomas, multiple myeloma, melanoma,glioma, glioblastomas, leukemia, sarcomas, leiomyomas, mesothelioma,GIST, and carcinomas of the lung, breast, ovary, cervix, liver, biliarytract, gastrointestinal tract, pancreas, prostate, and head and neck,wherein said method comprises administering to a patient in need thereofa compound of the present invention, or pharmaceutically acceptablecomposition thereof.

In certain embodiments, the present invention provides a method for thetreatment of conditions which include an accumulation of excessextracellular matrix; a fibrotic condition (which can be induced by drugor radiation), e.g., scleroderma, lupus nephritis, connective tissuedisease, wound healing, surgical scarring, spinal cord injury, CNSscarring, acute injury, pulmonary fibrosis (such as idiopathic pulmonaryfibrosis and radiation-induced pulmonary fibrosis), chronic obstructivepulmonary disease, adult respirator/distress syndrome, acute lunginjury, drug-induced lung injury, glomerulonephritis, diabeticnephropathy, hypertension-induced nephropathy, alimentary track orgastrointestinal fibrosis, renal fibrosis, hepatic or biliary fibrosis,liver cirrhosis, primary biliary cirrhosis, cirrhosis due to fatty liverdisease (alcoholic and nonalcoholic steatosis), primary sclerosingcholangitis, restenosis, cardiac fibrosis, opthalmic scarring,fibrosclerosis, fibrotic cancers, fibroids, fibroma, fibroadenomas,fibrosarcomas, transplant arteriopathy, and keloid); and, otherconditions such as cachexia, hypertension, ankylosing spondylitis,demyelination in multiple sclerosis, cerebral angiopathy and Alzheimer'sdisease, wherein said method comprises administering to a patient inneed thereof a compound of the present invention, or pharmaceuticallyacceptable composition thereof.

Other embodiments provide a method for treating a hematopoietic ornon-hematopoietic malignancy in a patient in need thereof, wherein saidmethod comprises administering to a patient in need thereof a compound,of the present invention, or pharmaceutically acceptable compositionthereof. In certain embodiments, the present invention provides a methodfor treating AML, chronic myelogenous leukemia (CML), mastocytosis,anaplastic large-cell lymphoma, ALL, gastrointestinal stromal tumor(GIST), T-cell lymphoma, adenoid cytsic carcinoma, angiosarcoma,endometrial carcinoma, small cell lung carcinoma, prostate cancer,ovarian cancer, breast carcinoma, thyroid carcinoma, malignant melanomaor colon carcinoma, wherein said method comprises administering to apatient in need thereof a compound of the present invention, orpharmaceutically acceptable composition thereof.

According to another aspect, the present invention provides a method fortreating a disease or condition selected from cancer such as braincancer, genitourinary tract cancer, lymphatic system cancer, stomachcancer, cancer of the larynx, lung cancer, pancreatic cancer, breastcancer, Kaposi's sarcoma, and leukemia; endometriosis, benign prostatichyperplasia; vascular diseases such as restenosis and atherosclerosis;autoimmune diseases such as rheumatoid arthritis and psoriasis; ocularconditions such as proliferative or angiogenic retinopathy and maculardegeneration; and inflammatory diseases such as contact dermatitis,asthma and delayed hypersensitivity reactions, wherein said methodcomprises administering to a patient in need thereof a compound of thepresent invention, or pharmaceutically acceptable composition thereof.

In certain embodiments, the present invention provides a method fortreating a disease or condition selected from osteoporosis, rheumatoidarthritis patients, inflammatory bowel disease, glomerulonephritis,allograft rejection, and arteriosclerosis, and cancer, wherein saidmethod comprises administering to a patient in need thereof a compoundof the present invention, or pharmaceutically acceptable compositionthereof.

A family of type III receptor tyrosine kinases including c-Kit plays animportant role in the maintenance, growth and development ofhematopoietic and non-hematopoietic cells. c-Kit regulates maintenanceof stem cell/early progenitor pools as well the development of maturelymphoid and myeloid cells. Upon activation, the kinase domain inducesautophosphorylation of the receptor as well as the phosphorylation ofvarious cytoplasmic proteins that help propogate the activation signalleading to growth, differentiation and survival. Some of the downstreamregulators of c-Kit receptor signaling include, PLCγ, PI3-kinase, Grb-2,SHIP and Src related kinases. Both receptor tyrosine kinases have beenshown to play a role in a variety of hematopoietic and non-hematopoieticmalignancies. Mutations that induce ligand independent activation ofc-Kit have been implicated acute-myelogenous leukemia (AML), acutelymphocytic leukemia (ALL), mastocytosis and gastrointestinal stromaltumor (GIST). These mutations include single amino acid changes in thekinase domain or internal tandem duplications, point mutations orin-frame deletions of the juxtamembrane region of the receptors. Inaddition to activating mutations, ligand dependent (autocrine orparacrine) stimulation of over-expressed wild-type c-Kit can contributeto the malignant phenotype.

According to another embodiment, the invention provides a method fortreating or lessening the severity of a cKit-mediated disease orcondition in a patient comprising the step of administering to saidpatient a composition according to the present invention.

The term “cKit-mediated disease” or “condition”, as used herein meansany disease or other deleterious condition in which cKit is known toplay a role. Accordingly, another embodiment of the present inventionrelates to treating or lessening the severity of one or more diseases inwhich cKit is known to play a role. Specifically, the present inventionrelates to a method of treating or lessening the severity of a diseaseor condition selected from a proliferative disorder, wherein said methodcomprises administering to a patient in need thereof a compositionaccording to the present invention.

The term “c-KIT-mediated disease”, as used herein means any disease orother deleterious condition in which a c-KIT family kinase is known toplay a role. Such conditions include, without limitation, AML, chronicmyelogenous leukemia (CML), mastocytosis, anaplastic large-celllymphoma, ALL, gastrointestinal stromal tumor (GIST), T-cell lymphoma,adenoid cytsic carcinoma, angiosarcoma, endometrial carcinoma, smallcell lung carcinoma, prostate cancer, ovarian cancer, breast carcinoma,thyroid carcinoma, malignant melanoma and colon carcinoma.

KDR is a tyrosine kinase receptor that also binds VEGF (vascularendothelial growth factor). The binding of VEGF to the KDR receptorleads to angiogenesis, which is the sprouting of capillaries frompreexisting blood vessels. High levels of VEGF are found in variouscancers causing tumor angiogenesis and permitting the rapid growth ofcancerous cells. Therefore, suppressing VEGF activity is a way toinhibit tumor growth, and it has been shown that this can be achieved byinhibiting KDR receptor tyrosine kinase. For example inhibitors of thetyrosine kinase are reported to also suppress tumor vascularization andthe growth of multiple tumors.

Examples of cancers that may be treated by such inhibitors include braincancer, genitourinary tract cancer, lymphatic system cancer, stomachcancer, cancer of the larynx, lung cancer, pancreatic cancer, breastcancer, Kaposi's sarcoma, and leukemia. Other diseases and conditionsassociated with abnormal tyrosine kinase activity include vasculardisease, autoimmune diseases, ocular conditions, and inflammatorydiseases.

According to another embodiment, the invention provides a method fortreating or lessening the severity of a KDR-mediated disease orcondition in a patient comprising the step of administering to saidpatient a composition according to the present invention.

The term “KDR-mediated disease”, as used herein means any disease orother deleterious condition in which a KDR family kinase is known toplay a role. Accordingly, another embodiment of the present inventionrelates to treating or lessening the severity of one or more diseases inwhich KDR is known to play a role. Specifically, the present inventionrelates to a method of treating or lessening the severity of a diseaseor condition selected from cancer such as brain cancer, genitourinarytract cancer, lymphatic system cancer, stomach cancer, cancer of thelarynx, lung cancer, pancreatic cancer, breast cancer, Kaposi's sarcoma,and leukemia; endometriosis, benign prostatic hyperplasia; vasculardiseases such as restenosis and atherosclerosis; autoimmune diseasessuch as rheumatoid arthritis and psoriasis; ocular conditions such asproliferative or angiogenic retinopathy and macular degeneration; andinflammatory diseases such as contact dermatitis, asthma and delayedhypersensitivity reactions.

cFMS is the receptor for Colony-stimulating factor 1 (CSF-1) whichpromotes the survival, proliferation, and differentiation of mononuclearphagocyte lineages. CSF-1 exerts its activities by binding tocell-surface cFMS receptors, resulting in autophosphorylation byreceptor cFMS kinase and a subsequent cascade of intracellular signals.Receptor expression in macrophage lineages is consistent with theability of exogenous CSF-1 to increase cytokine production in mice afterlipopolysaccharide (LPS) challenge, increase the production of monocytesand macrophages in mice, and exacerbate arthritis in mice and rats. Micewith a nonfunctional CSF-1 ligand or receptor are osteopetrotic,deficient in several macrophage populations, and have diminishedresponse to inflammatory challenge.

The CSF-1-cFMS-receptor pathway is up-regulated in a number of humanpathologies that involve chronic activation of tissue macrophagepopulations and, thus, could be a target for drug therapy. CSF-1promotes osteoclast development and bone degradation in vitro and, thus,could contribute to the excessive osteoclast activity in osteoporosisand at sites of orthopedic implant failure. CSF-1 is elevated in thesynovial fluid of rheumatoid arthritis patients, and synovialfibroblasts from rheumatoid arthritis patients produce high levels ofCSF-1, suggesting a role for CSF-1 in joint degradation. Increases inCSF-1 production are also associated with the accumulation of tissuemacrophages seen in inflammatory bowel disease, glomerulonephritis,allograft rejection, and arteriosclerosis. In addition, the growth ofseveral tumor types is associated with overexpression of CSF-1 and cFMSreceptor in cancer cells and/or tumor stroma.

The compounds and compositions, according to the method of the presentinvention, may be administered using any amount and any route ofadministration effective for treating or lessening the severity ofcancer, an autoimmune disorder, a neurodegenerative or neurologicaldisorder, schizophrenia, a bone-related disorder, liver disease, or acardiac disorder. The exact amount required will vary from subject tosubject, depending on the species, age, and general condition of thesubject, the severity of the infection, the particular agent, its modeof administration, and the like. The compounds of the invention arepreferably formulated in dosage unit form for ease of administration anduniformity of dosage. The expression “dosage unit form” as used hereinrefers to a physically discrete unit of agent appropriate for thepatient to be treated. It will be understood, however, that the totaldaily usage of the compounds and compositions of the present inventionwill be decided by the attending physician within the scope of soundmedical judgment. The specific effective dose level for any particularpatient or organism will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The term “patient”, as usedherein, means an animal, preferably a mammal, and most preferably ahuman.

The pharmaceutically acceptable compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), bucally, as an oral or nasal spray, orthe like, depending on the severity of the infection being treated. Incertain embodiments, the compounds of the invention may be administeredorally or parenterally at dosage levels of about 0.01 mg/kg to about 50mg/kg and preferably from about 1 mg/kg to about 25 mg/kg, of subjectbody weight per day, one or more times a day, to obtain the desiredtherapeutic effect.

Liquid dosage forms for oral administration include, but are not limitedto, pharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups and elixirs. In addition to the active compounds,the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a compound of the present invention,it is often desirable to slow the absorption of the compound fromsubcutaneous or intramuscular injection. This may be accomplished by theuse of a liquid suspension of crystalline or amorphous material withpoor water solubility. The rate of absorption of the compound thendepends upon its rate of dissolution that, in turn, may depend uponcrystal size and crystalline form. Alternatively, delayed absorption ofa parenterally administered compound form is accomplished by dissolvingor suspending the compound in an oil vehicle. Injectable depot forms aremade by forming microencapsule matrices of the compound in biodegradablepolymers such as polylactide-polyglycolide. Depending upon the ratio ofcompound to polymer and the nature of the particular polymer employed,the rate of compound release can be controlled. Examples of otherbiodegradable polymers include poly(orthoesters) and poly(anhydrides).Depot injectable formulations are also prepared by entrapping thecompound in liposomes or microemulsions that are compatible with bodytissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pills,and granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes. Solid compositions of a similartype may also be employed as fillers in soft and hard-filled gelatincapsules using such excipients as lactose or milk sugar as well as highmolecular weight polethylene glycols and the like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositions thatcan be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention. Additionally, the presentinvention contemplates the use of transdermal patches, which have theadded advantage of providing controlled delivery of a compound to thebody. Such dosage forms can be made by dissolving or dispensing thecompound in the proper medium. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate can becontrolled by either providing a rate controlling membrane or bydispersing the compound in a polymer matrix or gel.

According to one embodiment, the invention relates to a method ofinhibiting protein kinase activity in a biological sample comprising thestep of contacting said biological sample with a compound of thisinvention, or a composition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting PDGFR (alpha and beta), cKit, KDR, or cFMS activity in abiological sample comprising the step of contacting said biologicalsample with a compound of this invention, or a composition comprisingsaid compound. In certain embodiments, the invention relates to a methodof irreversibly inhibiting PDGFR (alpha and beta), cKit, KDR, or cFMSactivity in a biological sample comprising the step of contacting saidbiological sample with a compound of this invention, or a compositioncomprising said compound.

The term “biological sample”, as used herein, includes, withoutlimitation, cell cultures or extracts thereof; biopsied materialobtained from a mammal or extracts thereof; and blood, saliva, urine,feces, semen, tears, or other body fluids or extracts thereof.

Inhibition of protein kinase, or a protein kinase selected from PDGFR(alpha and beta), cKit, KDR, or cFMS, activity in a biological sample isuseful for a variety of purposes that are known to one of skill in theart. Examples of such purposes include, but are not limited to, bloodtransfusion, organ-transplantation, biological specimen storage, andbiological assays.

Another embodiment of the present invention relates to a method ofinhibiting protein kinase activity in a patient comprising the step ofadministering to said patient a compound of the present invention, or acomposition comprising said compound.

According to another embodiment, the invention relates to a method ofinhibiting one or more of PDGFR (alpha and beta), cKit, KDR, or cFMSactivity in a patient comprising the step of administering to saidpatient a compound of the present invention, or a composition comprisingsaid compound. According to certain embodiments, the invention relatesto a method of irreversibly inhibiting one or more of PDGFR (alpha andbeta), cKit, KDR, or cFMS activity in a patient comprising the step ofadministering to said patient a compound of the present invention, or acomposition comprising said compound. In other embodiments, the presentinvention provides a method for treating a disorder mediated by one ormore of PDGFR (alpha and beta), cKit, KDR, or cFMS, in a patient in needthereof, comprising the step of administering to said patient a compoundaccording to the present invention or pharmaceutically acceptablecomposition thereof. Such disorders are described in detail herein.

Depending upon the particular condition, or disease, to be treated,additional therapeutic agents, which are normally administered to treatthat condition, may also be present in the compositions of thisinvention. As used herein, additional therapeutic agents that arenormally administered to treat a particular disease, or condition, areknown as “appropriate for the disease, or condition, being treated”.

For example, compounds of the present invention, or a pharmaceuticallyacceptable composition thereof, are administered in combination withchemotherapeutic agents to treat proliferative diseases and cancer.Examples of known chemotherapeutic agents include, but are not limitedto, Adriamycin, dexamethasone, vincristine, cyclophosphamide,fluorouracil, topotecan, taxol, interferons, platinum derivatives,taxane (e.g., paclitaxel), vinca alkaloids (e.g., vinblastine),anthracyclines (e.g., doxorubicin), epipodophyllotoxins (e.g.,etoposide), cisplatin, methotrexate, actinomycin D, actinomycin D,dolastatin 10, colchicine, emetine, trimetrexate, metoprine,cyclosporine, daunorubicin, teniposide, amphotericin, alkylating agents(e.g., chlorambucil), 5-fluorouracil, camptothecin, cisplatin,metronidazole, and Gleevec™, among others. In other embodiments, acompound of the present invention is administered in combination with abiologic agent, such as Avastin or VECTIBIX.

In certain embodiments, compounds of the present invention, or apharmaceutically acceptable composition thereof, are administered incombination with an antiproliferative or chemotherapeutic agent selectedfrom any one or more of Abarelix, aldesleukin, Aldesleukin, Alemtuzumab,Alitretinoin, Allopurinol, Altretamine, Amifostine, Anastrozole, Arsenictrioxide, Asparaginase, Azacitidine, BCG Live, Bevacuzimab, Avastin,Fluorouracil, Bexarotene, Bleomycin, Bortezomib, Busulfan, Calusterone,Capecitabine, Camptothecin, Carboplatin, Carmustine, Celecoxib,Cetuximab, Chlorambucil, Cisplatin, Cladribine, Clofarabine,Cyclophosphamide, Cytarabine, Dactinomycin, Darbepoetin alfa,Daunorubicin, Denileukin, Dexrazoxane, Docetaxel, Doxorubicin (neutral),Doxorubicin hydrochloride, Dromostanolone Propionate, Epirubicin,Epoetin alfa, Erlotinib, Estramustine, Etoposide Phosphate, Etoposide,Exemestane, Filgrastim, floxuridine fludarabine, Fulvestrant, Gefitinib,Gemcitabine, Gemtuzumab, Goserelin Acetate, Histrelin Acetate,Hydroxyurea, Ibritumomab, Idarubicin, Ifosfamide, Imatinib Mesylate,Interferon Alfa-2a, Interferon Alfa-2b, Irinotecan, Lenalidomide,Letrozole, Leucovorin, Leuprolide Acetate, Levamisole, Lomustine,Megestrol Acetate, Melphalan, Mercaptopurine, 6-MP, Mesna, Methotrexate,Methoxsalen, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone,Nelarabine, Nofetumomab, Oprelvekin, Oxaliplatin, Paclitaxel,Palifermin, Pamidronate, Pegademase, Pegaspargase, Pegfilgrastim,Pemetrexed Disodium, Pentostatin, Pipobroman, Plicamycin, PorfimerSodium, Procarbazine, Quinacrine, Rasburicase, Rituximab, Sargramostim,Sorafenib, Streptozocin, Sunitinib Maleate, Talc, Tamoxifen,Temozolomide, Teniposide, VM-26, Testolactone, Thioguanine, 6-TG,Thiotepa, Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin,ATRA, Uracil Mustard, Valrubicin, Vinblastine, Vincristine, Vinorelbine,Zoledronate, or Zoledronic acid.

Other examples of agents the inhibitors of this invention may also becombined with include, without limitation: treatments for Alzheimer'sDisease such as Aricept® and Excelon®; treatments for Parkinson'sDisease such as L-DOPA/carbidopa, entacapone, ropinrole, pramipexole,bromocriptine, pergolide, trihexephendyl, and amantadine; agents fortreating Multiple Sclerosis (MS) such as beta interferon (e.g., Avonex®and Rebif®), Copaxone®, and mitoxantrone; treatments for asthma such asalbuterol and Singulair®; agents for treating schizophrenia such aszyprexa, risperdal, seroquel, and haloperidol; anti-inflammatory agentssuch as corticosteroids, TNF blockers, IL-1 RA, azathioprine,cyclophosphamide, and sulfasalazine; immunomodulatory andimmunosuppressive agents such as cyclosporin, tacrolimus, rapamycin,mycophenolate mofetil, interferons, corticosteroids, cyclophophamide,azathioprine, and sulfasalazine; neurotrophic factors such asacetylcholinesterase inhibitors, MAO inhibitors, interferons,anti-convulsants, ion channel blockers, riluzole, and anti-Parkinsonianagents; agents for treating cardiovascular disease such asbeta-blockers, ACE inhibitors, diuretics, nitrates, calcium channelblockers, and statins; agents for treating liver disease such ascorticosteroids, cholestyramine, interferons, and anti-viral agents;agents for treating blood disorders such as corticosteroids,anti-leukemic agents, and growth factors; and agents for treatingimmunodeficiency disorders such as gamma globulin.

Those additional agents may be administered separately from an inventivecompound-containing composition, as part of a multiple dosage regimen.Alternatively, those agents may be part of a single dosage form, mixedtogether with a compound of this invention in a single composition. Ifadministered as part of a multiple dosage regime, the two active agentsmay be submitted simultaneously, sequentially or within a period of timefrom one another normally within five hours from one another.

As used herein, the term “combination,” “combined,” and related termsrefers to the simultaneous or sequential administration of therapeuticagents in accordance with this invention. For example, a compound of thepresent invention may be administered with another therapeutic agentsimultaneously or sequentially in separate unit dosage forms or togetherin a single unit dosage form. Accordingly, the present inventionprovides a single unit dosage form comprising a compound of formula I,an additional therapeutic agent, and a pharmaceutically acceptablecarrier, adjuvant, or vehicle.

The amount of both, an inventive compound and additional therapeuticagent (in those compositions which comprise an additional therapeuticagent as described above) that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. Preferably,compositions of this invention should be formulated so that a dosage ofbetween 0.01-100 mg/kg body weight/day of an inventive can beadministered.

In those compositions which comprise an additional therapeutic agent,that additional therapeutic agent and the compound of this invention mayact synergistically. Therefore, the amount of additional therapeuticagent in such compositions will be less than that required in amonotherapy utilizing only that therapeutic agent. In such compositionsa dosage of between 0.01-100 μg/kg body weight/day of the additionaltherapeutic agent can be administered.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

The compounds of this invention, or pharmaceutical compositions thereof,may also be incorporated into compositions for coating an implantablemedical device, such as prostheses, artificial valves, vascular grafts,stents and catheters. Vascular stents, for example, have been used toovercome restenosis (re-narrowing of the vessel wall after injury).However, patients using stents or other implantable devices risk clotformation or platelet activation. These unwanted effects may beprevented or mitigated by pre-coating the device with a pharmaceuticallyacceptable composition comprising a kinase inhibitor. Implantabledevices coated with a compound of this invention are another embodimentof the present invention.

EXEMPLIFICATION

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

Example 1 Synthesis of Intermediate A Step 1:3-Dimethylamino-1-pyridin-3-yl-propenone

3-Acetylpyridine (2.5 g, 20.64 mmol) and N,N-dimethyl-formamidedimethylacetal (3.20 ml, 24 mmol) were refluxed in ethanol (10 mL)overnight. The reaction mixture was cooled to room temperature andevaporated under reduced pressure. Diethyl ether (20 mL) was added tothe residue and the mixture was cooled to 0° C. The mixture was filteredto give 3-dimethylamino-1-pyridin-3-yl-propenone (1.9 g, 10.78 mmol) asyellow crystals. (Yield: 52%.) This material was used in subsequentsteps without further purification.

Step 2: N-(2-Methyl-5-nitro-phenyl)-guanidinium nitrate

2-Methyl-5-nitro aniline (10 g, 65 mmol) was dissolved in ethanol (25mL), and concentrated HNO₃ (4.6 mL) was added to the solution dropwisefollowed by 50% aqueous solution of cyanamide (99 mmol). The reactionmixture was refluxed overnight and then cooled to 0° C. The mixture wasfiltered and the residue was washed with ethyl acetate and diethyl etherand dried to provide N-(2-Methyl-5-nitro-phenyl)-guanidinium nitrate(4.25 g, yield: 34%).

Step 3: 2-methyl-5-nitrophenyl-(4-pyridin-3-yl-pyrimidin-2-yl)-amine

To a suspension of 3-dimethylamino-1-pyridin-3-yl-propenone (1.70 g, 9.6mmol) and N-(2-methyl-5-nitro-phenyl)-guanidinium nitrate (2.47 g, 9.6mmol) in 2-propanol (20 mL) was added NaOH (430 mg, 10.75 mmol) and theresulting mixture was refluxed for 24 h. The reaction mixture was cooledto 0° C. and the resulting precipitate was filtered. The solid residuewas suspended in water and filtered and then washed with 2-propanol anddiethyl ether and dried. 0.87 g (2.83 mmol) of2-methyl-5-nitrophenyl-(4-pyridin-3-yl-pyrimidin-2-yl)-amine wasisolated. (Yield: 30%.)

Step 4: 4-Methyl-N-3-(4-pyridin-3-yl-pyrimidin-2-yl)-benzene-1,3-diamine(Intermediate A)

A solution of SnCl₂.2H₂O (2.14 g, 9.48 mmol) in concentratedhydrochloric acid (8 mL) was added to2-methyl-5-nitro-phenyl-(4-pyridin-3-yl-pyrimidin-2-yl)-amine (0.61 g,1.98 mmol) with vigorous stirring. After 30 min of stirring the mixturewas poured onto crushed ice, made alkaline with K₂CO₃, and extractedthree times with of ethyl acetate (50 ml). Organic phases were combined,dried over MgSO₄, and evaporated to dryness. Recrystallization fromdichloromethane resulted in 252.6 mg (0.91 mmol) of4-methyl-N-3-(4-pyridin-3-yl-pyrimidin-2-yl)-benzene-1,3-diamine (yield:46%) as an off-white solid.

Example 1

Step 1: 4-(acrylamido)benzoic acid

A solution of 4-aminobenzoic acid (1.40 g, 10 mmol) in DMF (10 mL) andpyridine (0.5 ml) was cooled to 0° C. To this solution was added ofacryloyl chloride (0.94 g, 10 mmol) and the resulting mixture wasstirred for 3 hours. The mixture was poured into 200 ml of water and thewhite solid obtained was filtered, washed with water and ether. Dryingunder high vacuum provided 1.8 g of the desired product which was usedin the next step without purification.

Step 2

4-(Acrylamido)benzoic acid (82 mg, 0.43 mmol) and Intermediate A (100mg, 0.36 mmol) were dissolved in pyridine (4 ml) under nitrogen andstirred. To this solution was added 1-propane phosphonic acid cyclicanhydride (0.28 g, 0.43 mmol) and the resulting solution was stirredovernight at room temperature. The solvent was evaporated to a smallvolume and then poured into a 50 ml of cold water. The solid formed wasfiltered and a yellow powder was obtained. Purification of the crudeproduct by column chromatography (95:5 CHCl₃:MeOH) provided 30 mg of4-acrylamido-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)benzamide(II-1) as a white powder. MS (M+H+): 251.2; ¹H NMR (DMSO-D₆, 300 MHz) δ(ppm): 10.42 (s, 1H), 10.11 (s, 1H), 9.26 (d, 1H, J=2.2 Hz), 8.99 (s,1H), 8.68 (dd, 1H, J=3.0 and 1.7 Hz), 8.51 (d, 1H, J=5.2 Hz), 8.48 (m,1H), 8.07 (d, 1H, J=1.7 Hz), 7.95 (d, 2H, J=8.8 Hz), 7.79 (d, 2H, J=8.8Hz), 7.45 (m, 3H), 7.19 (d, 1H, J=8.5 Hz), 6.47 (dd, 1H, J=16.7 and 9.6Hz), 6.30 (dd, H, J=16.7 and 1.9 Hz), 5.81 (dd, 1H, J=9.9 and 2.2 Hz),2.22 (s, 3H).

Example 2

Compound II-10 can be synthesized from4-(2,5-dioxo-2H-pyrrol-1(5H)-yl)benzoic acid and Intermediate A insubstantially the same manner as described in Example 1.4-Maleimidobenzoic acid is commercially available from multiple vendors.

Example 3

Step-1: 4-(Acryloylaminomethyl)-benzoic acid

To a stirred solution of 4-aminomethyl benzoic acid (5 g, 33.0 mmol) andpyridine (1.5 mL) in DMF (35 mL) was added acryloyl chloride (2.97 g,33.0 mmol) at 0° C. dropwise. The reaction mixture was allowed to cometo room temperature and stirred further for 4 days. After that it waspoured in water (100 mL) and stirred for 15 min and then extracted withEtOAc (3×150 mL). The combined organic extract was washed with brine (50mL), dried over Na₂SO₄ and concentrated under reduced pressure to givecrude product. Column chromatography (silica, 230-400, mixtures ofCHCl₃/MeOH) provided the desired product (0.7 g, 9.95%) as a white solidwhich was used for next step.

Step-2:4-(Acryloylaminomethyl)-N-[4-methyl-3-(4-pyridin-3-ylpyrimidin-2-ylamino)phenyl]benzamide(II-6)

To a solution of 4-(acryloylaminomethyl)-benzoic acid (75 mg, 0.37 mmol)in CH₃CN (4 mL) was added BOP reagent (245 mg, 0.55 mmol), followed byDIEA (150 mg, 1.1 mmol) and Intermediate A (100 mg, 0.37 mmol). Thereaction mixture was allowed to stir at rt for 16 h and then quenchedwith water (10 mL) and extracted with ethyl acetate (3×5 mL). Thecombined organic extract was washed with water (2 mL), brine (2 mL).Drying over Na₂SO₄ followed by concentration under reduced pressure gavecrude product which was purified by column chromatography (silica,230-400, mixtures of chloroform/methanol) to get II-6 (70 mg, 41%) as awhite solid. MS (M+H⁺): 464.5, 1H NMR (DMSO-d₆, 400 MHZ) δ (ppm): 2.20(s, 3H), 4.40-4.42 (m, 2H), 5.62 (dd, J=2.2 & 10 Hz, 1H), 6.12 (dd,J=2.2 & 17 Hz, 1H), 6.26 (dd, J=10 & 17 Hz, 1H), 7.19 (d, J=8.2 Hz, 1H),7.37-7. 54 (m, 5H), 7.90 (d, J=8.2 Hz, 2H), 8.06 (s, 1H), 8.47-8.50 (m,2H), 8.67-8.68 (m, 2H), 9.0 (s, 1H), 9.26 (s, 1H), 10.15 (s, 1H).

Example 4

Step-1: 4-[(3-carboxy acryloylamino)methyl]benzoic acid

To a solution of 4-aminobenzoic acid (1.54 g, 10.18 mmol) in acetic acid(20 mL) was added maleic anhydride (1 g, 10.10 mmol) and the reactionwas stirred at room temperature for 18 h. A white solid precipitated outwhich was filtered, washed with water (3×5 mL) and dried to yield thedesired product (2.3 g, 90%) as a white amorphous solid which was usedas such for the next step.

Step-2: 4-(2,5-Dioxo-2,5-dihydropyrrol-1-methyl)benzoic acid

To a stirred solution of 4-[(3-carboxy acryloylamino)methyl]benzoic acid(1 g, 4.0 mmol) in dioxan (20 mL) was added P₂O₅ (0.84 g, 6.0 mmol) andthe reaction mixture was refluxed for 18 h. Then the reaction mixturewas cooled and solvents were removed on a rotatory evaporator underreduced pressure. The residue was taken in ice-cold water (5 mL) and thesolution was extracted with EtOAc (3×50 mL). The combined EtOAc extractwas washed with brine (25 mL), dried over Na₂SO₄ and concentrated underreduced pressure to yield4-(2,5-dioxo-2,5-dihydropyrrol-1-methyl)benzoic acid (0.1 g, 10%) as alight cream colored solid which was used without further purification.

Step-3:4-(2,5-Dioxo-2,5-dihydropyrrol-1-ylmethyl)-N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)-phenyl]benzamide(II-8)

To a solution of 4-(2,5-dioxo-2,5-dihydropyrrol-1-methyl)benzoic acid(84 mg, 0.37 mmol) in DMF (2 mL) was added BOP reagent (240 mg, 0.54mmol) and the reaction was stirred for 10 min. To it was added DIEA (140mg, 1.08 mmol) and the reaction was further stirred for 10 min. Finally,Intermediate A (100 mg, 0.37 mmol) was added to it and reaction wasallowed to stir at rt for 16 h. Then the reaction mixture was dilutedwith water (15 mL) and extracted with EtOAc (3×15 mL). The combinedEtOAc extract was washed with brine (5 mL), dried (Na₂SO₄) andconcentrated under reduced pressure to get 100 mg of the title compoundas a crude solid. It was further purified by prep. HPLC to get pure II-8(30 mg, 16.6%) as a light brown solid. MS (M+H⁺): 490.1, 1H NMR(DMSO-d₆, 400 MHZ) δ (ppm): 2.20 (s, 3H), 4.66 (s, 2H), 7.10 (s, 2H),7.19 (d, J=8.4 Hz, 1H), 7.35 (d, J=8.34 Hz, 2H), 7.41-7.52 (m, 3H), 7.88(d, J=8.3 Hz, 2H), 8.05 (d, J=1.8 Hz, 1H), 8.45-8.50 (m, 2H), 8.66 (dd,J=1.5 & 4.7 Hz, 1H), 8.97 (s, 1H), 9.25 (s, 1H), 10.16 (s, 1H).

Example 5

Compound II-15 can be synthesized by treating compound II-1 (preparedaccording to Example 1) with dimethyldioxirane in acetone using aprotocol similar to that described in Bioorganic & Medicinal Chemistry10 (2002) 355-360.

Example 6

Step 1

4-(Allylsulfonamido)benzoic acid can be prepared by treating 4-aminobenzoic acid with commercially available prop-2-ene-1-sulfonyl chloridein a mixture of DMF and pyridine using the protocol in Example 1.

Step 2

4-(Allylsulfonamido)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)benzamidecan be prepared by treating Intermediate A and4-(allylsulfonamido)benzoic acid in a manner substantially similar tothat described in Example 1.

Step 3

Compound II-22 can be synthesized by treating4-(Allylsulfonamido)-N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)benzamidewith dimethyldioxirane in acetone using a protocol similar to thatdescribed in Bioorganic & Medicinal Chemistry 10 (2002) 355-360.

Example 7

Step 1

Ethenesulfonyl chloride can be prepared from commercially available2-chloroethanesulfonyl chloride in the presence of triethylamine using aprotocol substantially similar to that described in Canadian Journal ofChemistry (1984), 62(10), 1977-95.

Step 2

4-(Vinylsulfonamido)benzoic acid can be synthesized by treating4-(aminomethyl)benzoic acid with ethenesulfonyl chloride in a mixture ofDMF and pyridine in a manner substantially similar to that described inExample 1.

Step 3

Compound II-23 can be synthesized from 4-(vinylsulfonamido)benzoic acidand Intermediate A using the same reaction conditions as in Example 1.

Example 8

Step 1

2-(2-Tosylhydrazono)acetic acid can be prepared by treating glyoxalicacid with 4-methylbenzenesulfonohydrazide in a mixture of HCl and waterusing the protocol described in Organic Syntheses, 49, 22-7; 1969

Step 2

Treatment of commercially available tert-butyl 4-aminobenzoate with2-(2-tosylhydrazono)acetic acid in the presence of DCC in THF followedby deprotection of the carboxylic acid with trifluoroacetic acid canprovide 4-[[[(4-methylphenyl)sulfonyl]hydrazono]acetylamino]benzoic acid(protocol similar to that described Tetrahedron Letters, 33(38),5509-10; 1992)

Step 3

Coupling of 4-[[[(4-methylphenyl)sulfonyl]hydrazono]acetylamino]benzoicacid and Intermediate A in the presence of DCC in THF followed bytreatment with triethyl amine can provide compound II-24 (protocolsimilar to that described Tetrahedron Letters, 33(38), 5509-10; 1992)

Example 9

Step-1

To a stirred solution of 2,6-dichlorobenzoic acid (0.20 g, 1.05 mmol) inacetone (10 mL) was added bromo ester (0.25 g, 1.25 mmol) and thereaction was allowed to stir at rt for 16 h. It was then filtered,filtrate concentrated to get a residue which on trituration withpetroleum ether offered the corresponding diester as a white crystallinesolid. Filtration, followed by drying gave the diester (0.29 g, 91%) asa white solid which was used for next step.

Step-2

The diester (0.26 g, 0.85 mmol) was dissolved in CH₂Cl₂ (5.0 mL) and toit was added trifluoroacetic acid (0.39 g, 3.38 mmol) at 0° C. Thereaction mixture was allowed to come to rt and further stirred for 12 hat rt. It was then concentrated to the corresponding acid (0.2 g, 95.3%)as a white solid.

Step-3

To a stirred suspension of acid (0.15 g, 0.80 mmol) in CH₂Cl₂ (3.0 mL)was added DMF (1 drop) followed by SOCl₂ (0.14 g, 1.2 mmol). Thereaction mixture was heated at 45° C. for 3 h, cooled and concentratedunder reduced pressure to give the acid chloride which was used as suchfor the next step without further purifications.

Step-4

To a stirred suspension of the acid chloride (HCl salt, 0.1 g, 0.2 mmol)in CH₂Cl₂ (3.0 mL) was added Et₃N (0.12 g, 1.1 mmol) at 0° C. To it wasadded freshly prepared Intermediate B (0.33 g, 1.3 mmol, crude) and thereaction mixture was allowed to stir at 0° C. to 10° C. for 1.5 h. Aftercompletion of the reaction (TLC) the reaction was quenched with water (1mL) and extracted with CH₂Cl₂ (3×5 mL). The combined CH₂Cl₂ extract waswashed with water (2 mL), brine (2 mL), dried (Na₂SO₄) and concentratedunder reduced pressure to get crude II-26 which was purified further bypreparative HPLC to get pure II-26 (0.07 g, 48%) as a yellow solid.LC/MS (M+H⁺): 627, 1H NMR (CD₃OD, 400 MHZ) δ (ppm): 2.21 (s, 3H), 5.01(s, 2H), 7.19 (d, J=8.48 Hz, 1H), 7.42-7.64 (m, 6H), 7.73 (d, J=8.76 Hz,2H), 7.95 (d, J=8.76 Hz, 2H), 8.06 (d, J=1.8 Hz, 1H), 8.46-8.51 (m, 2H),8.67 (dd, J=1.2 & 4.68 Hz, 1H), 9.0 (s, 1H), 9.28 (s, 1H), 10.1 (s, 1H),10.52 (s, 1H).

Synthesis of Intermediate B

Step-1

To a solution of 4-aminobenzoic acid (5.0 g, 36.44 mmol) in1,4-dioxan/10% aq. NaHCO₃ soln (1:1, 20 mL) was added a cat. amount ofTBAB (0.58 g, 1.82 mmol) and the reaction mixture was stirred at 0° C.To it was added BOC anhydride (11.91 g, 54.6 mmol) and the reaction wasallowed to stir at rt for 22 h. The reaction mixture was then acidifiedwith 10% aq. citric acid soln. when 4N-Boc-aminobenzoic acidprecipitated out as a white solid. It was filtered, washed with water(100 mL), dried to get 4N-Boc-aminobenzoic acid (6.8 g, 79%) as a whitesolid, which was used for next step.

Step-2

To a solution of 4N-Boc-aminobenzoic acid (0.85 g, 3.6 mmol) in CH₂Cl₂(20 mL) was added EDCI (0.76 g, 3.97 mmol) and HOBT (0.54 g, 3.97 mmol).The reaction was stirred for 15 min and to it was added DIPEA (0.51 g,3.97 mmol) and Intermediate A (1.0 g, 3.6 mmol). The reaction wasfurther stirred at rt for 24 h and then diluted with EtOAc (100 mL),washed with water (2×25 mL), brine (10 mL) and dried over Na₂SO₄.Concentration under reduced pressure provided the corresponding amide asa crude solid which was crystallized (CH₂Cl₂/MeOH) to get the amide (0.4g, 22%) as a white solid.

Step-3

A solution of the amide (0.4 g, 0.8 mmol) in dioxan (10 mL) was cooledto 0° C. and purged with dry HCl for 10 min (reaction mixture turnsacidic) and allowed to stir for 16 h. After that the solvents wereremoved under reduced pressure to get Intermediate B, which was washedwith ether (10 mL) to get Intermediate B (0.28 g, 87%) as an orangesolid.

Example 10

Step 1

2-(Chlorocarbonyl)phenyl hypochlorothioite can be prepared from2,2′-dithiodibenzoic acid by treatment with thionyl chloride asdescribed in Synthetic Communications, 13(12), 977-83; 1983

Step 2

Treatment of glycine with 2-(chlorocarbonyl)phenyl hypochlorothioite inthe presence of pyridine can provide2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetic acid as described in Farmaco,44(9), 795-807; 1989.

Step 3

Coupling of 2-(3-oxobenzo[d]isothiazol-2(3H)-yl)acetic acid andIntermediate A in the presence of DCC and N-hydroxysuccinimde inDMF/CH₂Cl₂ can provide compound II-30 (protocol similar to thatdescribed in Synthesis, (17), 2647-2654; 2003).

Example 11

To a stirred mixture of Intermediate B (0.4 g, 1.01 mmol) and pyridinylsulfonyl chloride (0.23 g, 1.11 mmol) in NMP (5 mL) was added dry K₂CO₃(0.35 g, 2.52 mmol) and the reaction was heated at 85° C. for 18 h. Thereaction mixture was then cooled, diluted with CH₂Cl₂ (5 mL) and allowedto pass through a small bed of Celite®. The filtrate was concentrated,purified by chromatography (alumina, mixtures of CHCl₃ & MeOH) to getcrude II-40 which was further purified by preparative HPLC to get II-40(0.015 g, 2.6%) as a pale yellow solid. LC/MS (M−H⁺): 569.8, 1H NMR(DMSO-d₆, 400 MHZ) δ (ppm): 2.19 (s, 3H), 7.16 (d, J=8.28 Hz, 1H), 7.22(d, J=8.68 Hz, 2H), 7.38-7.41 (m, 2H), 7.49-7.52 (m, 1H), 7.77-7.82 (m,3H), 8.0 (s, 1H), 8.44-8.49 (m, 2H), 8.67 (dd, J=1.36 & 4.68 Hz, 1H),8.74 (d, J=5.28 Hz, 1H), 8.96 (s, 1H), 9.14 (s, 1H), 9.25 (d, J=1.36 Hz,1H), 10.06 (s, 1H), 11.32 (s, 1H)

Example 12

To a stirred solution of Intermediate B (0.40 g, 1.01 mmol) andpyridinylsulfonyl chloride (0.32 g, 1.5 mmol) in NMP was added dry K₂CO₃(0.56 g, 4.04 mmol) and the reaction was heated at 85° C. for 18 h. Thereaction was then cooled, diluted with CH₂Cl₂ (5 mL) and passed througha pad of Celite®. The filtrate was concentrated and purified bychromatography (neutral alumina, mixtures of CHCl₃ & MeOH) to get II-41which was further purified by preparative HPLC to afford II-41 (0.028 g,4.8%) as an off white solid. LC/MS (M+H⁺): 572.1, 1H NMR (MeOD, 400 MHZ)δ (ppm): 2.31 (s, 3H), 7.24-7.28 (m, 3H), 7.35-7.38 (m, 2H), 7.53-7.56(m, 1H), 7.59 (d, J=8.44 Hz, 1H), 7.86 (d, J=8.72 Hz, 2H), 8.14-8.18 (m,2H), 8.46 (d, J=5.24 Hz, 1H), 8.57-8.61 (m, 2H), 8.63 (d, J=1.56 Hz,1H), 9.30 (s, 1H)

Example 13

Step-1: 6-Acryloylaminonaphthalene-2-carboxylic acid

To a stirred solution of 2-Amino-6-naphthoic acid (0.3 g, 1.6 mmol) inDMF (2 mL) and pyridine (0.3 mL) was added at 0° C., acryloyl chloride(0.15 g, 1.6 mmol). The resulting mixture was allowed to come to rt andstirred further for 2 h. After that it was poured in water (5 mL) andthe solid separated was filtered and dried under reduced pressure to getthe desired product (160 mg, 41.5%) as a pale brown solid. This solidwas used in the next step without further purification.

Step-2: 6-Acryloylaminonaphthalene-2-carboxylicacid[4-methyl-3-(4-pyridin-3-yl pyrimidin-2-ylamino)phenyl]amide (II-44)

To a stirred solution of 6-acryloylaminonaphthalene-2-carboxylic acid(74 mg, 0.31 mmol) and Intermediate A (85 mg, 0.31 mmol) in DMF (2 mL)was added DIEA (0.08 mL). To it was added HATU (0.176 g, 0.47 mmol) andthe reaction mixture was allowed to stir at rt for 16 h. The reactionmixture was then diluted with water (5 mL) and EtOAc (10 mL) and passedthrough a small pad of Celite® to remove inorganics. The organic andaqueous phases were separated and the aqueous phase was again extractedwith EtOAc (2×10 mL). The combined organics were washed with brine (10mL), dried (Na₂SO₄) and then purified by flash chromatography (silica,230-400, mixtures of CHCl₃/MeOH) to get II-44 (33 mg, 21%) as a paleyellow solid. MS (M+H): 500.5, 1H NMR (DMSO-d₆, 400 MHZ) δ (ppm): 2.23(s, 3H), 5.82 (d, J=10 Hz, 1H), 6.32 (d, J=16 Hz, 1H), 6.51 (dd, J=10 &16 Hz, 1H), 7.22 (d, J=8.2 Hz, 1H), 7.43 (dd, J=0.8 & 5.2 Hz, 1H),7.50-7.54 (m, 2H), 7.73 (d, J=9.2 Hz, 1H), 7.93-8.0 (m, 2H), 8.04 (d,J=8.8 Hz, 1H), 8.12 (s, 1H), 8.48-8.68 (m, 4H), 8.67 (d, J=4.7 Hz, 1H),9.0 (s, 1H), 9.28 (d, J=1 Hz, 1H), 10.34 (s, 1H), 10.49 (s, 1H)

Example 14

Step 1

tert-Butyl6-((4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylatecan be synthesized by treating commercially available2-[[(1,1-dimethylethyl)oxy]carbonyl]-1,2,3,4-tetrahydro-6-isoquinolinecarboxylicacid and Intermediate A using the same reaction conditions as in Example1.

Step 2

Deprotection of tert-butyl6-((4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)carbamoyl)-3,4-dihydroisoquinoline-2(1H)-carboxylatewith HCl/dioxane followed by treatment with acryloyl chloride in amixture of DMF and pyridine using the protocol in step 2 of Example 1can provide compound II-46.

Step-1

To a stirred solution of the aniline ester (5 g, 30.27 mmol) in ethanol(12.5 mL) was added conc. HNO₃ (3 mL), followed by 50% aq. solution ofcyanamide (1.9 g, 46.0 mmol) at rt. The reaction mixture was heated at90° C. for 16 h and then cooled to 0° C. A solid precipitated out whichwas filtered, washed with ethyl acetate (10 mL), diethyl ether (10 mL),and dried to give the corresponding guanidine (4.8 g, 76.5%) as a lightpink solid which was used without further purifications.

Step-2

A stirred solution of 3-acetyl pyridine (10.0 g, 82.56 mmol) andN,N-dimethylformamide dimethyl acetal (12.8 g, 96.00 mmol) in ethanol(40 mL) was refluxed for 16 h. It was then cooled to rt and concentratedunder reduced pressure to get a crude mass. The residue was taken inether (10 mL), cooled to 0° C. and filtered to get the correspondingenamide (7.4 g, 50.8%) as a yellow crystalline solid.

Step-3

A stirred mixture of the guanidine derivative (2 g, 9.6 mmol), theenamide derivative (1.88 g, 10.7 mmol) and NaOH (0.44 g, 11.0 mmol) inethanol (27 mL) was refluxed at 90° C. for 48 h. The reaction mixturewas then cooled and concentrated under reduced pressure to get aresidue. The residue was taken in ethyl acetate (20 mL) and washed withwater (5 mL). The organic and aqueous layers were separated and treatedseparately to get the corresponding ester and Intermediate Crespectively. The aq. layer was cooled and acidified with 1.5 N HCl(pH˜3-4) when a white solid precipitated out. The precipitate wasfiltered, dried and excess water was removed by azeotropic distillationover toluene (2×10 mL) to get Intermediate C (0.5 g) as a pale yellowsolid. 1H NMR (DMSO-d₆, 400 MHz) δ (ppm): 2.32 (s, 3H), 7.36 (d, J=10.44Hz, 1H), 7.53 (d, J=6.84 Hz, 1H), 760-7.72 (m, 2H), 8.26 (s, 1H), 8.57(d, J=6.84 Hz, 1H), 8.64 (d, J=10.28 Hz, 1H), 8.70-8.78 (bs, 1H), 9.15(s, 1H), 9.35 (s, 1H). The organic extract was washed with brine (3 mL),dried (Na₂SO₄) and concentrated under reduced pressure to get the esterof Intermediate C as crude solid. It was further purified by columnchromatography (SiO₂, 60-120 mesh, MeOH/CHCl₃: 10/90) to get the esterof Intermediate C (0.54 g) as a yellow solid.

Step-1

To a stirring solution of the nitroaniline (0.15 g, 0.7 mmol) in THF(0.3 mL) was added Et₃N (0.11 mL, 0.73 mmol) and DMAP (0.05 g, 0.44mmol). To it was added BOC anhydride (0.33 mL, 1.52 mmol) and thereaction was allowed to reflux for 5 h. The reaction mixture was thencooled, diluted with THF (15 mL) and washed with brine (5 mL). Theorganic phase was separated, dried over Na₂SO₄, filtered andconcentrated under reduced pressure to get a crude mass. The crudeproduct was further purified by column chromatography (SiO₂, 230-400mesh, Hexane/EtOAc:8/2) to get corresponding di-Boc protected aniline(0.25 g, 88%) as a white crystalline solid which was taken for next stepwithout further purification.

Step-2

A solution of Boc protected aniline (0.25 g, 0.62 mmol) in MeOH (5 mL)was hydrogenated (H₂, 3 Kg) over 10% Pd/C (0.14 g, 0.13 mmol) at 20° C.for 12 h. The reaction mixture was passed through a short pad ofCelite®, concentrated under reduced pressure to get the correspondinganiline as an off-white solid (0.18 g, 77.6%). 1H NMR (CD₃OD, 400 MHz) δ(ppm): 1.36 (s, 18H), 6.84-6.87 (m, 1H), 6.95-6.97 (m, 2H).

Example 15

a) HATU, DIEA, CH₃CN, 85° C., 16 h, b) TFA/CH₂Cl₂, 0° C. to rt, 3 h, c)acryloyl chloride, pyridine, DMF, rt

Step-1

Coupling of Intermediate C with diboc protected aniline in the presenceof HATU, DIEA in acetonitrile can provide the corresponding amide

Step-2

Deprotection of the Boc groups to give Intermediate D can beaccomplished by treating the amide with TFA in methylene chloride at 0°C. and then warming up to room temperature.

Step 3

Acylation of Intermediate D with acryloyl chloride in a mixture ofpyridine and DMF can provide III-1 similar to a procedure used inExample 1.

Example 16

Compound III-5 can be prepared by treating Intermediate D with ethenesulfonyl chloride in a mixture of pyridine and DMF using a protocolsimilar to that in Example 7.

Example 17

Compound III-7 can be prepared by treating Intermediate D with acidchloride prepared in Step 3, Example 9 in a mixture of triethyl amineand methylene chloride using a protocol similar to that in Example 9.

Example 18

Compound III-8 can be synthesized by treating compound III-1 (preparedaccording to Example 15) with dimethyldioxirane in acetone using aprotocol similar to that described in Bioorganic & Medicinal Chemistry10 (2002) 355-360.

Example 19

Compound III-10 can be synthesized by treating Intermediate D withchloropyridinyl sulfonyl chloride using a protocol similar to thatdescribed in Example 11.

Example 20

Compound III-11 can be synthesized by treating Intermediate D withchloropyridinyl sulfonyl chloride using a protocol similar to thatdescribed in Example 12.

Example 21

To a stirred solution of trans-4-dimethylamino-2-butenoic acid (0.13 g,0.80 mmol) in acetonitrile (1.0 mL) was added oxalyl chloride (0.153 g,1.2 mmol) at 0° C. The reaction mixture was allowed to stir at 0° C. for30 minutes and then at room temperature for 2 h. Finally it was heatedat 45° C. for 5 min, and cooled to room temperature. This mixture wasadded to a solution of Intermediate D (0.075 g, 0.16 mmol) inN-methylpyrrolidone (1.0 mL) at 0° C. The reaction mixture was stirredat 0° C. to 10° C. for 2 h, was quenched with cold water (1 mL), and wasextracted with CHCl₃ (3×5 mL). The combined organic extract was washedsequentially with water (1 mL) and brine (1 mL), and then was dried overNa₂SO₄. Concentration under reduced pressure followed by purification bycolumn chromatography (SiO₂, 230-400 mesh, CHCl₃/MeOH, 95/5) gave III-6(0.02 g, 22%) as a light yellow solid. ¹H NMR (DMSO-d6) δ ppm: 2.18 (s,6H), 2.34 (s, 3H), 3.06 (d, J=5.6 Hz, 2H), 6.25-6.45 (m, 1H), 6.65-6.85(m, 1H), 7.43 (d, J=8.12 Hz, 1H), 7.47-7.52 (m, 3H), 7.74 (dd, J=1.76 &8.0 Hz, 1H), 8.06 (dd, J=2 & 8.76 Hz, 1H), 8.22 (d, J=2.68 Hz, 1H), 8.29(s, 1H), 8.43-8.45 (m, 1H), 8.54 (d, J=5.16 Hz, 1H), 8.68 (dd, J=1.64 &4.8 Hz, 1H), 9.16 (s, 1H), 9.27 (d, J=1.8 Hz, 1H), 9.62 (s, 1H), 10.48(s, 1H); LCMS m/e: 576.3 (M+1).

Example 22

To a stirred solution of Intermediate D (0.1 g, 0.22 mmol) in THF (10mL) at 0° C. was added Et₃N (0.033 g, 0.32 mmol) under N₂. Chloroacetylchloride (0.029 g, 0.26 mmol) was added dropwise with stirring and thereaction mixture was allowed to come to room temperature and was stirredfor 12 h. The reaction mixture was concentrated under reduced pressureto give a residue, which was taken in EtOAc (10 mL). This solution waswashed with water (2 mL) and the aqueous layer was again extracted withEtOAc (2×10 mL). The EtOAc fractions were combined and were washed withbrine (2 mL). Following drying over Na₂SO₄ and filtration the EtOAcsolution and was concentrated under reduced pressure to give cruderesidue, which was then purified by column chromatography (SiO₂, 60-120mesh, CHCl₃/MeOH:9/1) to give III-14 (50 mg, 43%) as a pale yellowsolid. 1H NMR (DMSO-d₆) δ ppm: 2.34 (s, 3H), 4.30 (s, 2H), 7.42-7.48 (m,4H), 7.73-7.75 (m, 1H), 8.05-8.10 (m, 1H), 8.24 (d, J=2.2 Hz, 1H), 8.29(s, 1H), 8.43 (d, J=8.04 Hz, 1H), 8.54 (d, J=5.16 Hz, 1H), 8.67 (dd,J=1.6 & 4.76 Hz, 1H), 9.16 (s, 1H), 9.26 (d, J=2.2 Hz, 1H), 9.89 (s,1H), 10.50 (s, 1H); LCMS: m/e 541.2 (M+1)

Example 23 PDGFR Inhibition Assay

Method A:

Compounds may also be assayed as inhibitors of PDGFR in a mannersubstantially similar to the method described in Roberts, et al.,“Antiangiogenic and Antitumor Activity of a Selective PDGFR TyrosineKinase Inhibitor, CP-673,451” Cancer Research 65, 957-966, Feb. 1, 2005.In this assay, a glutathione S-transferase-tagged kinase domainconstruct of the intracellular portion of the PDGFR-β (amino acids693-1,401, accession no. J03278) is expressed in Sf-9 cells (baculovirusexpression system, Invitrogen, Carlsbad, Calif.). Enzyme kinetics aredetermined by incubating the enzyme with increasing concentrations ofATP in phosphorylation buffer [50 mmol/L HEPES (pH 7.3), 125 mmol/LNaCl, 24 mmol/L MgCl₂ in Nunc Immuno MaxiSorp 96-well plates previouslycoated with 100 μL of 100 μg/mL poly-Glu-Tyr (4:1 ratio) diluted in PBS.After 10 minutes, the plates are washed (PBS, 0.1% Tween 20), incubatedwith anti-phosphotyrosine-horseradish peroxidase antibody, and dilutedin PBS, 0.05% Tween 20, 3% BSA for 30 minutes at room temperature. Theplates are washed as above and incubated with3,3′,5,5′-tetramethylbenzidine. The reaction is stopped by adding anequal volume of 0.09 N H₂SO₄. The phosphotyrosine-dependent signal isthen quantitated on a plate reader at 450 nm. For routine enzyme assays,the enzyme is incubated with 10 μmol/L (final) ATP in the presence ofcompound diluted in DMSO (1.6% v/v DMSO assay final) for 30 minutes atroom temperature in plates, as above, previously coated with 100 μL of6.25 μg/mL poly-Glu-Tyr. The remainder of the assay is carried out asabove, and IC₅₀ values are calculated as percent inhibition of control.Selectivity assays are done as described above using purifiedrecombinant enzyme (generated as described above) and ATP concentrationsat or up to 3× above the K_(m) for each enzyme.

Method B:

Compounds were assayed as inhibitors of PDGFR in a manner substantiallysimilar to the method described by Invitrogen Corp (InvitrogenCorporation, 1600 Faraday Avenue, Carlsbad, Calif., CA;http://www.invitrogen.com/downloads/Z-LYTE_Brochure_(—)1205.pdf) usingthe Z'-LYTE™ biochemical assay procedure or similar biochemical assay.The Z'-LYTE™ biochemical assay employs a fluorescence-based,coupled-enzyme format and is based on the differential sensitivity ofphosphorylated and non-phosphorylated peptides to proteolytic cleavage.

Compound I-1 was tested at 0.1 μM and 1 μM in duplicate. Compound I-1showed a mean inhibition of PDGFR-α of 76% at 1 μM and 29% at 0.1 μM.

Method C

Compounds may also be assayed as inhibitors of PDGFR alpha (h) in amanner substantially similar to the method described by Upstate(http://www.upstate.com/img/pdf/KP_Protocol_(—)121506.pdf). The proteinis recombinant human PDGFRα, residues 550-end, containing an N-terminalHis6-tag. Expressed by baculovirus in Sf21 insect cells. Purified usingNi2+/NTA agarose. In a final reaction volume of 25 μL, PDGFR alpha(wild-type) (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA,10 mM MnCl₂, 0.1 mg/mL poly(Glu, Tyr) 4:1, 10 mM MgAcetate and[©-33P-ATP] (specific activity approx. 500 cpm/pmol, concentration asrequired). The reaction is initiated by the addition of the MgATP mix.After incubation for 40 minutes at room temperature, the reaction isstopped by the addition of 5 μL of a 3% phosphoric acid solution. 10 μLof the reaction is then spotted onto a Filtermat A and washed threetimes for 5 minutes in 75 mM phosphoric acid and once in methanol priorto drying and scintillation counting. The compound is dissolved to 50×final assay concentration in 100% DMSO, and assayed using 0.5 μL of thiscompound solution in a final reaction volume of 25 ul as above. DMSOonly (0.5 ul) is tested in parallel as a control

Method D

Briefly, 10× stock of PDGFRα (PV3811) enzyme, 1.13×ATP (AS001A) andY12-Sox peptide substrates (KCZ1001) was prepared in 1× kinase reactionbuffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl₂, 1 mM EGTA, 5 mMβ-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT(DS001A). 5 μL of enzyme were pre-incubated in a Corning (#3574)384-well, white, non-binding surface microtiter plate (Corning, N.Y.)for 30 min at 27° C. with a 0.5 μL volume of 50% DMSO and seriallydiluted compounds prepared in 50% DMSO. Kinase reactions were startedwith the addition of 45 μL of the ATP/Y9 or Y12-Sox peptide substratemix and monitored every 30-9 seconds for 60 minutes atλ_(ex)360/λ_(em)485 in a Synergy⁴ plate reader from BioTek (Winooski,Vt.). At the conclusion of each assay, progress curves from each wellwere examined for linear reaction kinetics and fit statistics (R², 95%confidence interval, absolute sum of squares). Initial velocity (0minutes to 20+ minutes) from each reaction was determined from the slopeof a plot of relative fluorescence units vs time (minutes) and thenplotted against inhibitor concentration to estimate IC₅₀ from log[Inhibitor] vs Response, Variable Slope model in GraphPad Prism fromGraphPad Software (San Diego, Calif.).

[PDGFRα]=2-5 nM, [ATP]=60 μM and [Y9-Sox peptide]=10 μM (ATP K_(Mapp)=61μM)

PDGFR inhibition data for certain compounds of the present invention areset forth in Table 5, below.

TABLE 5 PDGFR Inhibition Data Compound # % Inhibition Conc. (μM) IC₅₀(nM) II-1 76 1 172.94 II-2 91 1 2.2 II-4 28 1 — II-6 — — 29.8 II-8 — —10.69 II-23 — — 18.8 II-45 — — 7.9 II-52 — — 1017 II-53 — — 16.4 II-54 —— 184.8 II-55 — — 1.4 III-6 — — 5.90 III-14 — — 0.73

Example 24 PDGFR Mass Spectral Analysis of Compound I-1

Mass spectral analysis of PDGFR-α in the presence of test compound I-1was performed. PDGFR-α protein (supplied from Invitrogen: PV3811) wasincubated with 1 μM, 10 μM, and 100 μM test compound for 60 minutes.Specifically, 1 μL of 0.4 μg/μL PDGFR-α (Invitrogen PV3811) stocksolution (50 mM Tris HCl ph 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.02% TritonX-100, 2 mM DTT, 50% glycerol) was added to 9 μL of test compound in 10%DMSO (final concentration of 1 μM, 10 μM, and 100 μM). After 60 minutes,9 μL of 50 mM ammonium bicarbonate, 3.3 μL of 6 mM iodoacetamide in 50mM ammonium bicarbonate, and 1 μL of 35 ng/μL trypsin was added to stopthe reaction.

The tryptic digest was analyzed by mass spectrometer (MALDI-TOF) at 10μM. Of the five cysteine residues found in the PDGFR-α protein, four ofthe cysteine residues were identified as being modified byiodoacetamide, while the fifth cysteine residue was modified by the testcompound. Mass spectral analysis of the tryptic digests was consistentwith test compound being covalently bound to PDGFR-α protein at Cys814.MS/MS analysis of the tryptic digests confirmed presence of the testcompound at Cys814.

Example 25 PDGFR Mass Spectral Analysis of Compound III-14

Mass spectral analysis of PDGFR-α in the presence of test III-14 wasperformed. PDGFR-α (43 pmols) was incubated with compound III-14 (434pmols) for 3 hours at 10× access prior to tryptic digestion.Iodoacetamide was used as the alkylating agent after compoundincubation. For tryptic digests a 5 μl aliquot (7 pmols) was dilutedwith 10 μl of 0.1% TFA prior to micro C18 Zip Tipping directly onto theMALDI target using alpha cyano-4-hydroxy cinnamic acid as the matrix (5mg/ml in 0.1% TFA:Acetonitrile 50:50).

The tryptic digest was analyzed by mass spectrometer (MALDI-TOF). Massspectral analysis of the tryptic digests was consistent with testcompound being covalently bound to PDGFR-α protein at Cys814. MS/MSanalysis of the tryptic digests confirmed presence of the test compoundat Cys814.

Example 26 EOL-1 Cellular Proliferation Assay

EOL-1 cells purchased from DSMZ (ACC 386) were maintained in RPMI(Invitrogen #21870)+10% FBS+1% penicillin/streptomycin (Invitrogen#15140-122). For cell proliferation assays, cells in complete media wereplated in 96 well plates at a density of 2×10⁴ cells/well and incubatedin duplicate with compound ranging from 500 nM to 10 pM for 72 hours.Cell proliferation was assayed by measuring metabolic activity withAlamar Blue reagent (Invitrogen cat #DAL1100). After 8 hours incubationwith Alamar Blue at 37° C., absorbance was read at 590 nm and the IC₅₀of cellular proliferation was calculated using GraphPad. Dose responseinhibition of cell proliferation of EOL-1 cells with reference compoundand compound II-2 is depicted in FIG. 1.

Example 27 EOL-1 Cell Washout Assay

EOL-1 cells were grown in suspension in complete media and compound wasadded to 2×10⁶ cells per sample for 1 hour. After 1 hour, the cells werepelleted, the media was removed and replaced with compound-free media.Cells were washed every 2 hours and resuspended in fresh compound-freemedia. Cells were collected at specified timepoints, lysed in CellExtraction Buffer and 15 μg total protein lysate was loaded in eachlane. PDGFR phosphorylation was assay by western blot with Santa Cruzantibody sc-12910. The results of this experiment are depicted in FIG. 2where it is shown that relative to DMSO control and to a reversiblereference compound, compound II-2 maintained enzyme inhibition of PDGFRin EOL-1 cells after “washout” after 0 hours and 4 hours.

Example 28 cKit Inhibition Assay

Method A

Compounds may also be assayed as inhibitors of CKit in a mannersubstantially similar to the method described by Upstate(http://www.upstate.com/img/pdf/KP_Protocol_(—)121506.pdf). In thisassay, N-terminal GST tagged, recombinant, human Kit, amino acids544-end, expressed by baculovirus, in Sf21 insect cells and purifiedusing glutathione agarose is used. In a final reaction volume of 25 μL,c-kit (wild-type) (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mMEDTA, 10 mM MnCl₂, 0.1 mg/mL poly(Glu, Tyr) 4:1, 10 mM MgAcetate and[©-33P-ATP] (specific activity approx. 500 cpm/pmol, concentration asrequired). The reaction is initiated by the addition of the MgATP mix.After incubation for 40 minutes at room temperature, the reaction isstopped by the addition of 5 μL of a 3% phosphoric acid solution. 10 μLof the reaction is then spotted onto a Filtermat A and washed threetimes for 5 minutes in 75 mM phosphoric acid and once in methanol priorto drying and scintillation counting. The compound is dissolved to 50×final assay concentration in 100% DMSO, and assayed using 0.5 ul of thiscompound solution in a final reaction volume of 25 ul as above. DMSOonly (0.5 ul) is tested in parallel as a control.

Method B

Compounds may also be assayed as inhibitors of cKit in a mannersubstantially similar to the method described by CellSignal(http://www.cellsignal.com/pdf/7755.pdf). The GST-c-Kit fusion proteinis produced using a baculovirus expression system with a constructexpressing a fragment of human c-Kit (Thr544-Val976) with anamino-terminal GST tag. The protein is purified by one-step affinitychromatography using glutathione-agarose. In this assay 10 μl 10 mM ATPis added to 1.25 ml 6 μM KDR (Tyr996) Biotinylated Peptide substratepeptide. The mixture is diluted with dH20 to 2.5 ml to make2×ATP/substrate cocktail ([ATP]=40 μM, [substrate]=3 μm) The c-KITenzyme is transferred immediately from −80° C. to ice. The enzyme isallowed to thaw on ice. The vial is microcentrifuged briefly at 4° C. tobring liquid to the bottom of the vial and it is then returnedimmediately to ice. 10 μl of DTT (1.25 M) is added to 2.5 ml of4×HTScan™ Tyrosine Kinase Buffer (Cellsignal; 240 mM HEPES pH 7.5, 20 mMMgCl₂, 20 mM MnCl₂, 12 μM Na3VO4) to make DTT/Kinase buffer. 1.25 ml ofDTT/Kinase buffer is transferred to enzyme tube to make 4× reactioncocktail ([enzyme]=4 ng/μL in 4× reaction cocktail). 12.5 μl of the 4×reaction cocktail is incubated with 12.5 μl/well of prediluted compoundfor 5 minutes at room temperature. 25 μl of 2×ATP/substrate cocktail isadded to 25 μl/well preincubated reaction cocktail/compound. Thereaction plate is incubated at room temperature for 30 minutes and 50μl/well Stop Buffer (50 mM EDTA, pH 8) is added to stop the reaction. 25μl of each reaction and 75 μl dH2O/well is transferred to a 96-wellstreptavidin coated plate and incubated at room temperature for 60minutes. This is washed three times with 200 μl/well PBS/T. The primaryantibody, Phospho-Tyrosine mAb (P-Tyr-100), is diluted 1:1000 in PBS/Twith 1% BSA. 100 μl/well primary antibody is added and incubated at roomtemperature for 60 minutes. This is washed three times with 200 μl/wellPBS/T. Europium labeled anti-mouse IgG 1:500 in PBS/T is diluted with 1%BSA. 100 μl/well diluted antibody is then added. This is then incubatedat room temperature for 30 minutes and washed five times with 200μl/well PBS/T. 100 μl/well DELFIA® Enhancement Solution is added andthen incubated at room temperature for 5 minutes. A Time-Resolved PlateReader is used to detect 615 nm fluorescence emission. Compounds showinggreater than 50% inhibition versus standard wells containing the assaymixture and DMSO without test compound are titrated to determine IC₅₀values.

Method C

Compounds of the invention were assayed as inhibitors of c-KIT usinghuman recombinant c-KIT (obtained from Millipore, catalog number 14-559)and monitoring the phosphorylation of a fluorescein-labeled peptidesubstrate (1.5 μM). Reactions were carried out in 100 mM HEPES (pH 7.5),10 mM MnCl₂, 1 mM DDT, 0.015% Brij-35, and 300 μM ATP, with and withouttest compound. The reaction was started by adding the ATP and incubatingfor 1 hour at room temperature. The reaction was terminated by theaddition of stop buffer containing 100 mM HEPES (pH 7.5), 30 mM EDTA,0.015% Brij-35, and 5% DMSO. Phosphorylated and unphosphorylatedsubstrate was separated by charge using electrophoretic mobility shift.Product formed was compared to control wells to determine inhibition orenhancement of enzyme activity.

c-KIT inhibition data for certain compounds of the present invention areset forth in Table 6, below.

TABLE 6 c-KIT Inhibition Data Compound # % Inhibition Concentration (μM)II-1 82 1 II-2 88 1 II-5 66 1 II-6 91 1 II-8 78 1 II-26 94 1 II-44 95 1Method Dc-Kit (V654A and T6701) Omnia Assays for Potency Assessment withPre-Activated Enzyme:

Compounds were also tested as inhibitors of mutant c-KIT using theprotocol below which describes a continuous-read kinase assay to measureinherent potency of compounds against pre-activated c-KIT (V654A) andc-KIT (T670I) enzymes.

Briefly, 10× stocks of c-KIT (V654A) from Millipore (14-733) or c-Kit(T670I) from Cell Signaling (7922) plus 1 mM ATP (AS001A) and1.13×Y9-Sox or Y12-Sox peptide substrates (KCZ1001) were prepared in 1×kinase reaction buffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl₂, 1mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock, KB002A) and0.2 mM DTT (DS001A). 10× enzyme/ATP stocks were pre-incubated for 30 minat room temperature to pre-activate each enzyme prior to compoundexposure. 5 μL of each enzyme were then pre-incubated in a Corning(#3574) 384-well, white, non-binding surface microtiter plate (Corning,N.Y.) for 30 min at 27° C. with a 0.5 μL volume of 50% DMSO and seriallydiluted compounds prepared in 50% DMSO. Kinase reactions were startedwith the addition of 45 μL of the Y9 or Y12-Sox peptide substrate andmonitored every 30-90 seconds for 60 minutes at λ_(ex)360/λ_(em)485 in aSynergy⁴ plate reader from BioTek (Winooski, Vt.). At the conclusion ofeach assay, progress curves from each well were examined for linearreaction kinetics and fit statistics (R², 95% confidence interval,absolute sum of squares). Initial velocity (0 minutes to 20+ minutes)from each reaction was determined from the slope of a plot of relativefluorescence units vs time (minutes) and then plotted against inhibitorconcentration to estimate IC₅₀ from log [Inhibitor] vs Response,Variable Slope model in GraphPad Prism from GraphPad Software (SanDiego, Calif.).

[c-Kit V654A]=5 nM, [ATP]=100 μM and [Y9Sox]=10 μM (ATP K_(Mapp)=240 μM)

[c-Kit T670I]=5 nM, [ATP]=100 μM and [Y12-Sox]=10 μM (ATP K_(Mapp)=220μM)

Method E

c-Kit (V654A and T6701) Omnia Assays for Potency Assessment:

Briefly, 10× stocks of c-Kit (V654A) from Millipore (14-733) or c-Kit(T670I) from Cell Signaling (7922), 1.13×ATP (AS001A) and Y9-Sox orY12-Sox peptide substrates (KCZ1001) were prepared in 1× kinase reactionbuffer consisting of 20 mM Tris, pH 7.5, 5 mM MgCl₂, 1 mM EGTA, 5 mMβ-glycerophosphate, 5% glycerol (10× stock, KB002A) and 0.2 mM DTT(DS001A). 5 μL of each enzyme were pre-incubated in a Corning (#3574)384-well, white, non-binding surface microtiter plate (Corning, N.Y.)for 30 min at 27° C. with a 0.5 μL volume of 50% DMSO and seriallydiluted compounds prepared in 50% DMSO. Kinase reactions were startedwith the addition of 45 μL of the Y9 or Y12-Sox peptide substrate andmonitored every 30-90 seconds for 60 minutes at λ_(ex)360/λ_(em)485 in aSynergy⁴ plate reader from BioTek (Winooski, Vt.). At the conclusion ofeach assay, progress curves from each well were examined for linearreaction kinetics and fit statistics (R², 95% confidence interval,absolute sum of squares). Initial velocity (0 minutes to 20+ minutes)from each reaction was determined from the slope of a plot of relativefluorescence units vs time (minutes) and then plotted against inhibitorconcentration to estimate IC₅₀ from log [Inhibitor] vs Response,Variable Slope model in GraphPad Prism from GraphPad Software (SanDiego, Calif.).

[c-Kit V654A]=5 nM, [ATP]=220 μM and [Y9-Sox]=10 μM (ATP K_(Mapp)=240μM)

[c-Kit T670I]=5 nM, [ATP]=220 μM and [Y12-Sox]=10 μM (ATP K_(Mapp)=220μM)

Mutant c-KIT (V654A) inhibition data for exemplary compounds aresummarized in Table 7, below, where IC₅₀ values were obtained using theabove-described assay protocol Method D and IC₅₀ Apparent values wereobtained using the above-described assay protocol Method E.

TABLE 7 Mutant c-KIT (V654A) Inhibition Data Compound # IC₅₀ (nM) IC₅₀Apparent (nM) II-2 — 83 II-40 10000 — II-41 — 173 II-53 937 260 II-5410000 — II-55 9151 — III-6 3989 216 III-14 2011 72

Mutant c-KIT (T6701) inhibition data for exemplary compounds aresummarized in Table 8, below.

TABLE 8 Mutant c-KIT (T6701) Inhibition Data Compound # IC₅₀ (nM) IC₅₀Apparent (nM) III-6 — 1566 III-14 72.4 205

Example 29 c-KIT Mass Spectral Analysis of Compound III-14

Mass spectral analysis of c-KIT in the presence of test compound III-14was performed. Specifically, c-KIT kinase (86 pmols) was incubated withcompound III-14 (863 pmols) for 3 hours at 10× access prior to trypticdigestion. Iodoacetamide was used as the alkylating agent after compoundincubation. A newer sample was also prepared that used a higher grade oftrypsin which eliminated the chymotryptic activity. For tryptic digestsa 5 μl aliquot (14 pmols) was diluted with 10 ul of 0.1% TFA prior tomicro C18 Zip Tipping directly onto the MALDI target using alphacyano-4-hydroxy cinnamic acid as the matrix (5 mg/ml in 0.1%TFA:acetonitrile 50:50).

The tryptic digest was analyzed by mass spectrometer (MALDI-TOF). Massspectral analysis of the tryptic digests was consistent with testcompound being covalently bound to c-KIT protein at two target cysteinresidues: Cys808 (minor) and Cys788 (major).

Example 30 cKIT Washout Assay

GIST430 cells were seeded in a 6 well plate at a density of 8×10⁵cells/well and treated with 1 μM compound diluted in complete media for90 minutes the next day. After 90 minutes, the media was removed andcells were washed with compound-free media. Cells were washed every 2hours and resuspended in fresh compound-free media. Cells were collectedat specified time-points, lysed in Cell Extraction Buffer (InvitrogenFNN0011) supplemented with Roche complete protease inhibitor tablets(Roche 11697498001) and phosphatase inhibitors (Roche 04 906 837 001)and 10 μg total protein lysate was loaded in each lane. c-KITphosphorylation was assayed by western blot with pTyr (4G10) antibodyand total kit antibody from Cell Signaling Technology. The results ofthis experiment are depicted in FIG. 3 where it is shown that compoundIII-14 maintains c-KIT enzyme inhibition in GIST430 cells after“washout” at 0 hours and 6 hours.

Example 31 cFMS Inhibition Assay

Compounds may also be assayed as inhibitors of cFMS (h) in a mannersubstantially similar to the method described by Upstate(http://www.upstate.com/img/pdf/KP_Protocol_(—)121506.pdf). The proteinis expressed as a N-terminal His-tagged, recombinant, human cFms, aminoacids 538-end, in baculovirus in Sf21 insect cells and purified usingNi2+/NTA-agarose. In a final reaction volume of 25 μL, Fms (h) (5-10 mU)is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 250 μM KKKSPGEYVNIEFG,10 mM MgAcetate and [γ-33P-ATP] (specific activity approx. 500 cpm/pmol,concentration as required). The reaction is initiated by the addition ofthe MgATP mix. After incubation for 40 minutes at room temperature, thereaction is stopped by the addition of 5 μL of a 3% phosphoric acidsolution. 10 μL of the reaction is then spotted onto a P30 filtermat andwashed three times for 5 minutes in 75 mM phosphoric acid and once inmethanol prior to drying and scintillation counting. The compound isdissolved to 50× final assay concentration in 100% DMSO, and assayedusing 0.5 μl of this compound solution in a final reaction volume of 25ul as above. DMSO only (0.5 μl) is tested in parallel as a control

Example 32 KDR Inhibition Assay

Method A

Compounds may be screened for their ability to inhibit KDR using astandard coupled enzyme assay (Fox et al., Protein Sci., (1998) 7,2249). Assays are carried out in a mixture of 200 mM HEPES 7.5, 10 mMMgCl₂, 25 mM NaCl, 1 mM DTT and 1.5% DMSO. Final substrateconcentrations in the assay are 300 μM ATP (Sigma Chemicals) and 10 μMpoly E4Y (Sigma). Assays are carried out at 37° C. and 30 nM KDR. Finalconcentrations of the components of the coupled enzyme system are 2.5 mMphosphoenolpyruvate, 200 μM NADH, 30 μg/ML pyruvate kinase and 10 μg/mllactate dehydrogenase.

An assay stock buffer solution is prepared containing all of thereagents listed above, with the exception of ATP and the test compoundof interest. 177 μl of the stock solution is placed in a 96 well platefollowed by addition of 3 μl of 2 mM DMSO stock containing the testcompound (final compound concentration 30 μM). The plate is preincubatedfor about 10 minutes at 37° C. and the reaction initiated by addition of20 μA of ATP (final concentration 300 μM). Rates of reaction areobtained using a Molecular Devices plate reader (Sunnyvale, Calif.) overa 5 minute read time at 37° C. Compounds showing greater than 50%inhibition versus standard wells containing the assay mixture and DMSOwithout test compound are titrated to determine IC₅₀ values determined.

Method B

Compounds may also be assayed as inhibitors of KDR (h) in a mannersubstantially similar to the method described by Upstate(http://www.upstate.com/img/pdf/KP_Protocol_(—)121506.pdf). The proteinis expressed as a N-terminal 6His-tagged, recombinant, human KDR aminoacids 790-end, expressed by baculovirus in Sf21 insect cells andpurified using Ni²⁺/NTA agarose. In a final reaction volume of 25 μL,KDR (h) (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 250μM KKKSPGEYVNIEFG, 10 mM MgAcetate and [γ-33P-ATP] (specific activityapprox. 500 cpm/pmol, concentration as required). The reaction isinitiated by the addition of the MgATP mix. After incubation for 40minutes at room temperature, the reaction is stopped by the addition of5 μL of a 3% phosphoric acid solution. 10 μL of the reaction is thenspotted onto a P30 filtermat and washed three times for 5 minutes in 75mM phosphoric acid and once in methanol prior to drying andscintillation counting. The compound is dissolved to 50× final assayconcentration in 100% DMSO, and assayed using 0.5 μl of this compoundsolution in a final reaction volume of 25 ul as above. DMSO only (0.5ul) is tested in parallel as a control.

Method C

Compounds were assayed as inhibitors of KDR in a manner substantiallysimilar to the method described by Invitrogen. A kinase reaction bufferwas prepared by dilution of the 5× Kinase Buffer stock solution(available from Invitrogen, item number PV3189) by adding 4 mL of 5×stock to 16 mL H₂O to make 20 mL of 1× kinase reaction buffer.

Kinase reactions were performed in a 10 μL volume in low-volume 384-wellplates. Typically, Corning model 3676 (black) or 3673 (white) plates areused. The concentration of substrate (Fluorescein-Poly GAT orFluorescein-Poly GT, available from Invitrogen) in the assay was 200 nM,and the 1× kinase reaction buffer consists of 50 mM HEPES pH 7.5, 0.01%BRIJ-35, 10 mM MgCl₂, and 1 mM EGTA. The 2×KDR (VEGFR2)/Tyr 01 PeptideMixture was prepared in 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl₂,1 mM EGTA. The final 10 μL Kinase Reaction consisted of 0.5-11.7 ng KDR(VEGFR2) and 2 μM Tyr 01 Peptide in 50 mM HEPES pH 7.5, 0.01% BRIJ-35,10 mM MgCl₂, 1 mM EGTA. The kinase reaction was allowed to proceed for 1hour at room temperature before a 5 μL of a 1:128 dilution ofDevelopment Reagent B was added.

Compound II-2 was tested as an inhibitor of KDR using the above assayprotocol (Method C) resulting in 7% inhibition of the KDR enzyme at aconcentration of 10 μM of test compound.

Method D

Briefly, 10× stocks of KDR from Invitrogen or BPS Bioscience (PV3660 or40301), 1.13×ATP (AS001A) and Y9-Sox peptide substrate (KCZ1001) wereprepared in 1× kinase reaction buffer consisting of 20 mM Tris, pH 7.5,5 mM MgCl₂, 1 mM EGTA, 5 mM β-glycerophosphate, 5% glycerol (10× stock,KB002A) and 0.2 mM DTT (DS001A). 5 μL of enzyme were pre-incubated in aCorning (#3574) 384-well, white, non-binding surface microtiter plate(Corning, N.Y.) for 30 min at 27° C. with a 0.5 μL volume of 50% DMSOand serially diluted compounds prepared in 50% DMSO. Kinase reactionswere started with the addition of 45 μL of the ATP/Y9 or Y12-Sox peptidesubstrate mix and monitored every 30-9 seconds for 60 minutes atλ_(ex)360/λ_(em)485 in a Synergy⁴ plate reader from BioTek (Winooski,Vt.). At the conclusion of each assay, progress curves from each wellwere examined for linear reaction kinetics and fit statistics (R², 95%confidence interval, absolute sum of squares). Initial velocity (0minutes to 20+ minutes) from each reaction was determined from the slopeof a plot of relative fluorescence units vs time (minutes) and thenplotted against inhibitor concentration to estimate IC₅₀ from log[Inhibitor] vs Response, Variable Slope model in GraphPad Prism fromGraphPad Software (San Diego, Calif.).

[KDR]=2.5 nM, [ATP]=75 μM and [Y12-Sox peptide]=5 μM (ATP K_(Mapp)=75μM)

KDR inhibition data, obtained using above assay protocol Method D, forexemplary compounds are summarized in Table 9, below.

TABLE 9 KDR Inhibition Data Compound # IC₅₀ (μM) II-2 3.0 II-6 10.0II-23 2.2 II-45 10.0 II-52 3.0 II-55 4.5 III-6 8.1 III-14 2.1

While we have described a number of embodiments of this invention, it isapparent that our basic examples may be altered to provide otherembodiments that utilize the compounds and methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the appended claims rather than by the specificembodiments that have been represented by way of example.

We claim:
 1. A method for treating a PDGFR-, c-KIT-, KDR, or cFMS-mediated disorder in a patient in need thereof, comprising the step of administering to said patient compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: T is —NHC(O)— or —C(O)NH—; W is CH or N; each of R^(a), R^(b), R^(c), and R^(d) is independently selected from R, OR, or halogen; each R is independently hydrogen, lower alkyl, or lower haloalkyl; R¹ is -L-Y, wherein: L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain wherein L has at least one double bond and at least one methylene unit of L is replaced by cyclopropylene, —NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, —N═N—, or —C(═N₂)—; and one or two additional methylene units of L are optionally and independently replaced by —O—, —N(R)—, or —C(O)—; or L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain wherein L has at least one triple bond and one or two additional methylene units of L are optionally and independently replaced by —NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, —O—, —N(R)—, or —C(O)—; or L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain wherein one methylene unit of L is replaced by cyclopropylene and one or two additional methylene units of L are independently replaced by —C(O)—, —NRC(O)—, —C(O)NR—, —N(R)SO₂—, or —SO₂N(R)—; or L is a bivalent C₁₋₈, straight or branched, alkylene chain, wherein at least one methylene unit of L is replaced by —C(═N₂)—, and one or two additional methylene units of L are optionally and independently replaced by —NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, —O—, —N(R)—, or —C(O)—; and Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo, halogen, or CN, or a 3-10 membered monocyclic or bicyclic, saturated, partially unsaturated, or aryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and wherein said ring is substituted with 1-4 groups independently selected from -Q-Z, oxo, halogen, CN, or C₁₋₆ aliphatic, wherein: Q is a covalent bond or a bivalent C₁₋₆ saturated or unsaturated, straight or branched, hydrocarbon chain, wherein one or two methylene units of Q are optionally and independently replaced by —NR—, —NRC(O)—, —C(O)NR—, —S—, —O—, —C(O)—, —SO—, or —SO₂—; and Z is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo, halogen, or CN; or L is a covalent bond or a bivalent C₁₋₈ saturated or unsaturated, straight or branched, hydrocarbon chain, wherein one, two, or three methylene units of L are optionally and independently replaced by —NR—, —N(R)C(O)—, —C(O)N(R)—, —O—, —C(O)—, —OC(O)—, —C(O)O—, —S—, —SO—, —SO₂—, or —C(═S)—; and Y is selected from:

R² is selected from R, halogen, —N(R)C(O)OR, or 1-imidazoyl substituted with R, or: R¹ and R² are taken together with their intervening atoms to form a 5-7 membered saturated, partially unsaturated, or aryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, wherein said ring is substituted with -L-Y and 0-3 groups independently selected from oxo, halogen, CN, or C₁₋₆ aliphatic; and R³ is selected from hydrogen, lower alkyl, or halogen, or a composition thereof, wherein the PDGFR-, c-KIT-, KDR, or cFMS-mediated disorder is selected from breast cancer and rheumatoid arthritis.
 2. The method according to claim 1, wherein the compound of formula I irreversibly inhibits PDGFR, c-KIT, KDR, or cFMS, by covalently modifying Cys814 of PDGFR-α, Cys822 of PDGFR-β, Cys1024 of KDR, Cys788 of c-KIT, or Cys774 of cFMS.
 3. The method according to claim 1, wherein the disorder is breast cancer.
 4. The method according to claim 1, wherein the disorder is rheumatoid arthritis.
 5. The method according to claim 1, wherein said compound is of formula II-a or III-a:

or a pharmaceutically acceptable salt thereof, wherein: L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain wherein L has at least one double bond and at least one methylene unit of L is replaced by cyclopropylene, —NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, —N═N—, or —C(═N₂)—, and one or two additional methylene units of L are optionally and independently replaced by —O—, —N(R)—, or —C(O)—; and R² is selected from R, halogen, —N(R)C(O)OR, or 1-imidazoyl substituted with R.
 6. The method according to claim 5, wherein: Y is hydrogen or C₁₋₆ aliphatic optionally substituted with oxo, halogen, or CN; and R³ is lower alkyl.
 7. The method according to claim 6, wherein L is —NHC(O)CH═CH—, —NHC(O)CH═CHCH₂N(CH₃)—, —NHC(O)CH═CHCH₂O—, —CH₂NHC(O)CH═CH—, —NHSO₂CH═CH—, —NHSO₂CH═CHCH₂—, —NHC(O)(C═N₂)—, —NHC(O)(C═N₂)C(O)—, or —NHC(O)C(═CH₂)CH₂—.
 8. The method according to claim 1, wherein: L is a bivalent C₂₋₈ straight or branched, hydrocarbon chain wherein L has at least one triple bond and one or two additional methylene units of L are optionally and independently replaced by —NRC(O)—, —C(O)NR—, —S—, —S(O)—, —SO₂—, —C(═S)—, —C(═NR)—, —O—, —N(R)—, or —C(O)—; and Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo, halogen, or CN, or a 3-10 membered monocyclic or bicyclic, saturated, partially unsaturated, or aryl ring having 0-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and wherein said ring is substituted with at 1-4 groups independently selected from -Q-Z, oxo, halogen, CN, or C₁₋₆ aliphatic.
 9. The method according to claim 8, wherein: L is —C≡C—, —C≡CCH₂N(isopropyl)-, —NHC(O)C≡CCH₂CH₂—, —CH₂—C≡C—CH₂—, or —C≡CCH₂O—; and Y is hydrogen, C₁₋₆ aliphatic optionally substituted with oxo, halogen, or CN, phenyl, pyridyl, or an optionally substituted saturated 3-6 membered monocyclic ring having 1-3 heteroatoms independently selected from nitrogen, oxygen, or sulfur, and wherein said ring is substituted with at 1-4 groups independently selected from -Q-Z, oxo, halogen, CN, or C₁₋₆ aliphatic.
 10. The method according to claim 1, wherein: L is a covalent bond, —CH₂—, —NH—, —CH₂NH—, —NHCH₂—, —NHC(O)—, —NHC(O)CH₂OC(O)—, —CH₂NHC(O)—, —NHSO₂—, —NHSO₂CH₂—, —NHC(O)CH₂OC(O)—, or —SO₂NH— and Y is selected from:


11. The method according to claim 1, wherein said compound is selected from the group consisting of:


12. The method according to claim 1 wherein said compound is selected from the group consisting of: 